Nucleosides and nucleotides with 3&#39;-hydroxy blocking groups

ABSTRACT

Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with acetal or thiocarbamate 3′-OH blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and the uses of fully functionalized nucleotides containing the 3′-OH blocking group for sequencing applications.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Application Nos. 62/784,970 and 62/784,994, both filed Dec.26, 2018 and are incorporated by reference in their entireties.

BACKGROUND Field

The present disclosure generally relates to nucleotides, nucleosides, oroligonucleotides comprising 3′-hydroxy protecting groups and their usein polynucleotide sequencing methods. Methods of preparing the3′-hydroxy protected nucleotides, nucleosides, or oligonucleotides arealso disclosed.

Description of the Related Art

Advances in the study of molecules have been led, in part, byimprovement in technologies used to characterize the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis and the study of hybridization events.

An example of the technologies that have improved the study of nucleicacids is the development of fabricated arrays of immobilized nucleicacids. These arrays consist typically of a high-density matrix ofpolynucleotides immobilized onto a solid support material. See, e.g.,Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways ofassembling the nucleic acids using a chemically sensitized glass surfaceprotected by a mask, but exposed at defined areas to allow attachment ofsuitably modified nucleotide phosphoramidites. Fabricated arrays canalso be manufactured by the technique of “spotting” knownpolynucleotides onto a solid support at predetermined positions (e.g.,Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

One way of determining the nucleotide sequence of a nucleic acid boundto an array is called “sequencing by synthesis” or “SBS”. This techniquefor determining the sequence of DNA ideally requires the controlled(i.e., one at a time) incorporation of the correct complementarynucleotide opposite the nucleic acid being sequenced. This allows foraccurate sequencing by adding nucleotides in multiple cycles as eachnucleotide residue is sequenced one at a time, thus preventing anuncontrolled series of incorporations from occurring. The incorporatednucleotide is read using an appropriate label attached thereto beforeremoval of the label moiety and the subsequent next round of sequencing.

In order to ensure that only a single incorporation occurs, a structuralmodification (“protecting group” or “blocking group”) is included ineach labeled nucleotide that is added to the growing chain to ensurethat only one nucleotide is incorporated. After the nucleotide with theprotecting group has been added, the protecting group is then removed,under reaction conditions which do not interfere with the integrity ofthe DNA being sequenced. The sequencing cycle can then continue with theincorporation of the next protected, labeled nucleotide.

To be useful in DNA sequencing, nucleotides, which are usuallynucleotide triphosphates, generally require a 3′-hydroxy protectinggroup so as to prevent the polymerase used to incorporate it into apolynucleotide chain from continuing to replicate once the base on thenucleotide is added. There are many limitations on the types of groupsthat can be added onto a nucleotide and still be suitable. Theprotecting group should prevent additional nucleotide molecules frombeing added to the polynucleotide chain whilst simultaneously beingeasily removable from the sugar moiety without causing damage to thepolynucleotide chain. Furthermore, the modified nucleotide needs to becompatible with the polymerase or another appropriate enzyme used toincorporate it into the polynucleotide chain. The ideal protecting groupmust therefore exhibit long-term stability, be efficiently incorporatedby the polymerase enzyme, cause blocking of secondary or furthernucleotide incorporation, and have the ability to be removed under mildconditions that do not cause damage to the polynucleotide structure,preferably under aqueous conditions.

Reversible protecting groups have been described previously. Forexample, Metzker et al., (Nucleic Acids Research, 22 (20): 4259-4267,1994) discloses the synthesis and use of eight 3′-modified2-deoxyribonucleoside 5′-triphosphates (3′-modified dNTPs) and testingin two DNA template assays for incorporation activity. WO 2002/029003describes a sequencing method which may include the use of an allylprotecting group to cap the 3′—OH group on a growing strand of DNA in apolymerase reaction.

In addition, the development of a number of reversible protecting groupsand methods of deprotecting them under DNA compatible conditions waspreviously reported in International Application Publication Nos. WO2004/018497 and WO 2014/139596, each of which is hereby incorporated byreference in its entirety.

SUMMARY

Some embodiments of the present disclosure relate to a nucleotide ornucleoside comprising a ribose or deoxyribose having a removable 3′-OHprotecting or blocking group forming a structure

covalently attached to the 3′-carbon atom, wherein:

each R^(1a) and R^(1b) is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, cyano, halogen, optionally substitutedphenyl, or optionally substituted aralkyl;

each R^(2a) and R^(2b) is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl,cyano, or halogen;

alternatively, R^(1a) and R^(2a) together with the atoms to which theyare attached form an optionally substituted five to eight memberedheterocyclyl group;

R³ is H, optionally substituted C₂-C₆ alkenyl, optionally substitutedC₃-C₇ cycloalkenyl, optionally substituted C₂-C₆ alkynyl, or optionallysubstituted (C₁-C₆ alkylene)Si(R⁴)₃; and

each R⁴ is independently H, C₁-C₆ alkyl, or optionally substitutedC₆-C₁₀ aryl. In some embodiments, when each R^(1a) and R^(1b) is H orC₁-C₆ alkyl, both R^(2a) and R^(2b) are H, then R³ is substituted C₂-C₆alkenyl, optionally substituted C₃-C₇ cycloalkenyl, optionallysubstituted C₂-C₆ alkynyl, or optionally substituted (C₁-C₆alkylene)Si(R⁴)₃. In some embodiments, when each R^(1a), R^(1b), R^(2a)and R^(2b) is H, then R³ is not H.

Some embodiments of the present disclosure relate to a nucleoside ornucleotide comprising a ribose or deoxyribose having a removable 3′-OHblocking group forming a structure

covalently attached to the 3′-carbon atom, wherein:

each of R⁵ and R⁶ is independently H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₈ alkoxyalkyl, optionally substituted—(CH₂)_(m)-phenyl, optionally substituted —(CH₂)_(n)-(5 or 6 memberedheteroaryl), optionally substituted —(CH₂)_(k)—C₃-C₇ carbocyclyl, oroptionally substituted —(CH₂)_(p)-(3 to 7 membered heterocyclyl);

each of —(CH₂)_(m)—, —(CH₂)_(n)—, —(CH₂)_(k)—, and —(CH₂)_(p)— isoptionally substituted; and

each of m, n, k, and p is independently 0, 1, 2, 3, or 4.

Some embodiments of the present disclosure relate to an oligonucleotideor polynucleotide comprising a 3′-OH blocked nucleotide moleculedescribed herein.

Some embodiments of the present disclosure relate to a method ofpreparing a growing polynucleotide complementary to a targetsingle-stranded polynucleotide in a sequencing reaction, comprisingincorporating a nucleotide molecule described herein into the growingcomplementary polynucleotide, wherein the incorporation of thenucleotide prevents the introduction of any subsequent nucleotide intothe growing complementary polynucleotide. In some embodiments, theincorporation of the nucleotide is accomplished by a polymerase, aterminal deoxynucleotidyl transferase (TdT), or a reverse transcriptase.In one embodiment, the incorporation is accomplished by a polymerase(e.g., a DNA polymerase).

Some further embodiments of the present disclosure relate to a methodfor determining the sequence of a target single-stranded polynucleotide,comprising:

(a) incorporating a nucleotide comprising a 3′-OH blocking group and adetectable label as described herein into a copy polynucleotide strandcomplementary to at least a portion of the target polynucleotide strand;

(b) detecting the identity of the nucleotide incorporated into the copypolynucleotide strand; and

(c) chemically removing the label and the 3′-OH blocking group from thenucleotide incorporated into the copy polynucleotide strand.

In some embodiments, the sequencing method further comprises (d) washingthe chemically removed label and the 3′ blocking group away from thecopy polynucleotide strand. In some embodiment, such washing step alsoremoves the unincorporated nucleotides. In some such embodiments, the 3′blocking group and the detectable label of the incorporated nucleotideare removed prior to introducing the next complementary nucleotide. Insome further embodiments, the 3′ blocking group and the detectable labelare removed in a single step of chemical reaction. In some embodiments,the sequential incorporation described herein is performed at least 50times, at least 100 times, at least 150 times, at least 200 times, or atleast 250 times.

Some further embodiments of the present disclosure relate to kitscomprising a plurality of nucleotide or nucleoside molecules describedherein, and packaging materials therefor. The nucleotides, nucleosides,oligonucleotides, or kits that are set forth herein may be used todetect, measure, or identify a biological system (including, forexample, processes or components thereof). Exemplary techniques that canemploy the nucleotides, oligonucleotides, or kits include sequencing,expression analysis, hybridization analysis, genetic analysis, RNAanalysis, cellular assay (e.g., cell binding or cell function analysis),or protein assay (e.g., protein binding assay or protein activityassay). The use may be on an automated instrument for carrying out aparticular technique, such as an automated sequencing instrument. Thesequencing instrument may contain two or more lasers operating atdifferent wavelengths to distinguish between different detectablelabels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line chart illustrating the stability of various 3′ blockednucleotides as a function of time in a buffer solution at 65° C.

FIG. 2A is a line chart illustrating the percentage (%) of remainingnucleotide (starting material) as a function of time comparing thedeblocking rate of nucleotide with 3′-AOM blocking group to nucleotidewith the 3′-O-azidomethyl (—CH₂N₃) blocking group in solution.

FIG. 2B is a line chart illustrating the percentage (%) of 3′ deblockednucleotides as a function of time comparing the deblocking rate of 3′blocked nucleotides with various acetal blocking groups in solution.

FIGS. 3A and 3B illustrate the sequencing results on Illumina MiniSeq®instrument using fully functionalized nucleotides (ffNs) with 3′-AOMblocking group in the incorporation mix.

FIG. 3C illustrates the sequencing error rate using fully functionalizednucleotides (ffNs) with 3′-AOM blocking group in the incorporation mixas compared to the standard ffNs with 3′-O-azidometyl blocking group.

FIGS. 4A and 4B each illustrates comparison of the primary sequencingmetrics including phasing, pre-phasing and error rate using fullyfunctionalized nucleotides with 3′-AOM and 3′-O-azidomethyl blockinggroups using two different DNA polymerases (Pol 812 and Pol 1901)respectively.

FIG. 5 is a line chart illustrating the sequencing stability of fullyfunctionalized nucleotides with 3′-AOM or 3′-O-azidomethyl blockinggroups as a function of time in a buffer solution at 45° C.

FIG. 6 is a line chart illustrating the stability of nucleosides withvarious 3′ blocking groups as a function of time in a buffer solution at65° C.

FIG. 7 is a line chart illustrating the percentage (%) of remaining 3′blocked nucleotide as a function of time, comparing the cleavage(deblocking) rate of a thiocarbamate 3′ blocking groupdimethylthiocarbamate (DMTC) under two different conditions (Oxone® orNaIO₄) to that of the 3′-O-azidomethyl (3′-O—CH₂N₃) blocking group.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to nucleosides andnucleotides with 3′-OH acetal or thiocarbamate blocking groups forsequencing applications, for example, sequencing-by-synthesis (SBS).These blocking groups offer better stability in solution compared tothose known in the art. In particular, the 3′-OH blocking groups haveimproved stability during the synthesis of the fully functionalizednucleotides (ffNs) and also great stability in solution duringformulation, storage and operation on the sequencing instruments. Inaddition, the 3′-OH blocking groups described herein may also achievelow pre-phasing, lower signal decay for improved data quality, whichenables longer reads from the sequencing applications.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. The use of the term “including” as well as other forms, suchas “include”, “includes,” and “included,” is not limiting. The use ofthe term “having” as well as other forms, such as “have”, “has,” and“had,” is not limiting. As used in this specification, whether in atransitional phrase or in the body of the claim, the terms “comprise(s)”and “comprising” are to be interpreted as having an open-ended meaning.That is, the above terms are to be interpreted synonymously with thephrases “having at least” or “including at least.” For example, whenused in the context of a process, the term “comprising” means that theprocess includes at least the recited steps, but may include additionalsteps. When used in the context of a compound, composition, or device,the term “comprising” means that the compound, composition, or deviceincludes at least the recited features or components, but may alsoinclude additional features or components.

As used herein, common organic abbreviations are defined as follows:

° C. Temperature in degrees Centigrade

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

dGTP Deoxyguanosine triphosphate

dTTP Deoxythymidine triphosphate

ddNTP Dideoxynucleotide triphosphate

ffN Fully functionalized nucleotide

RT Room temperature

SBS Sequencing by Synthesis

SM Starting material

As used herein, the term “array” refers to a population of differentprobe molecules that are attached to one or more substrates such thatthe different probe molecules can be differentiated from each otheraccording to relative location. An array can include different probemolecules that are each located at a different addressable location on asubstrate. Alternatively, or additionally, an array can include separatesubstrates each bearing a different probe molecule, wherein thedifferent probe molecules can be identified according to the locationsof the substrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid. Exemplaryarrays in which separate substrates are located on a surface include,without limitation, those including beads in wells as described, forexample, in U.S. Pat. No. 6,355,431 B1, US 2002/0102578 and PCTPublication No. WO 00/63437. Exemplary formats that can be used in theinvention to distinguish beads in a liquid array, for example, using amicrofluidic device, such as a fluorescent activated cell sorter (FACS),are described, for example, in U.S. Pat. No. 6,524,793. Further examplesof arrays that can be used in the invention include, without limitation,those described in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071;5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269;6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413;6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO95/11995; WO 95/35505; EP 742 287; and EP 799 897.

As used herein, the term “covalently attached” or “covalently bonded”refers to the forming of a chemical bonding that is characterized by thesharing of pairs of electrons between atoms. For example, a covalentlyattached polymer coating refers to a polymer coating that forms chemicalbonds with a functionalized surface of a substrate, as compared toattachment to the surface via other means, for example, adhesion orelectrostatic interaction. It will be appreciated that polymers that areattached covalently to a surface can also be bonded via means inaddition to covalent attachment.

As used herein, any “R” group(s) represent substituents that can beattached to the indicated atom. An R group may be substituted orunsubstituted. If two “R” groups are described as “together with theatoms to which they are attached” forming a ring or ring system, itmeans that the collective unit of the atoms, intervening bonds and thetwo R groups are the recited ring. For example, when the followingsubstructure is present:

and R¹ and R² are defined as selected from the group consisting ofhydrogen and alkyl, or R¹ and R² together with the atoms to which theyare attached form an aryl or carbocyclyl, it is meant that R¹ and R² canbe selected from hydrogen or alkyl, or alternatively, the substructurehas structure:

where A is an aryl ring or a carbocyclyl containing the depicted doublebond.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “C_(a) to C_(b)” in which “a” and “b” are integers referto the number of carbon atoms in an alkyl, alkenyl or alkynyl group, orthe number of ring atoms of a cycloalkyl or aryl group. That is, thealkyl, the alkenyl, the alkynyl, the ring of the cycloalkyl, and ring ofthe aryl can contain from “a” to “b”, inclusive, carbon atoms. Forexample, a “C₁ to C₄ alkyl” group refers to all alkyl groups having from1 to 4 carbons, that is, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—,CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—; a C₃ to C₄ cycloalkyl grouprefers to all cycloalkyl groups having from 3 to 4 carbon atoms, thatis, cyclopropyl and cyclobutyl. Similarly, a “4 to 6 memberedheterocyclyl” group refers to all heterocyclyl groups with 4 to 6 totalring atoms, for example, azetidine, oxetane, oxazoline, pyrrolidine,piperidine, piperazine, morpholine, and the like. If no “a” and “b” aredesignated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl, oraryl group, the broadest range described in these definitions is to beassumed. As used herein, the term “C₁-C₆” includes C₁, C₂, C₃, C₄, C₅and C₆, and a range defined by any of the two numbers. For example,C₁-C₆ alkyl includes C₁, C₂, C₃, C₄, C₅ and C₆ alkyl, C₂-C₆ alkyl, C₁-C₃alkyl, etc. Similarly, C₂-C₆ alkenyl includes C₂, C₃, C₄, C₅ and C₆alkenyl, C₂-C₅ alkenyl, C₃-C₄ alkenyl, etc.; and C₂-C₆ alkynyl includesC₂, C₃, C₄, C₅ and C₆ alkynyl, C₂-C₅ alkynyl, C₃-C₄ alkynyl, etc. C₃-C₈cycloalkyl each includes hydrocarbon ring containing 3, 4, 5, 6, 7 and 8carbon atoms, or a range defined by any of the two numbers, such asC₃-C₇ cycloalkyl or C₅-C₆ cycloalkyl.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group may bedesignated as “C₁-C₄alkyl” or similar designations. By way of exampleonly, “C₁-C₆ alkyl” indicates that there are one to six carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C₁-C₉ alkoxy”, including but not limitedto methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 6carbon atoms. The alkenyl group may be designated as “C₂-C₆ alkenyl” orsimilar designations. By way of example only, “C₂-C₆ alkenyl” indicatesthat there are two to six carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groupsinclude, but are in no way limited to, ethenyl, propenyl, butenyl,pentenyl, and hexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 6carbon atoms. The alkynyl group may be designated as “C₂-C₆ alkynyl” orsimilar designations. By way of example only, “C₂-C₆ alkynyl” indicatesthat there are two to six carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

As used herein, “heteroalkyl” refers to a straight or branchedhydrocarbon chain containing one or more heteroatoms, that is, anelement other than carbon, including but not limited to, nitrogen,oxygen and sulfur, in the chain backbone. The heteroalkyl group may have1 to 20 carbon atoms, although the present definition also covers theoccurrence of the term “heteroalkyl” where no numerical range isdesignated. The heteroalkyl group may also be a medium size heteroalkylhaving 1 to 9 carbon atoms. The heteroalkyl group could also be a lowerheteroalkyl having 1 to 6 carbon atoms. The heteroalkyl group may bedesignated as “C₁-C₆ heteroalkyl” or similar designations. Theheteroalkyl group may contain one or more heteroatoms. By way of exampleonly, “C₄-C₆ heteroalkyl” indicates that there are four to six carbonatoms in the heteroalkyl chain and additionally one or more heteroatomsin the backbone of the chain.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e.,two or more fused rings that share two adjacent carbon atoms) containingonly carbon in the ring backbone. When the aryl is a ring system, everyring in the system is aromatic. The aryl group may have 6 to 18 carbonatoms, although the present definition also covers the occurrence of theterm “aryl” where no numerical range is designated. In some embodiments,the aryl group has 6 to 10 carbon atoms. The aryl group may bedesignated as “C₆-C₁₀ aryl,” “C₆ or C₁₀ aryl,” or similar designations.Examples of aryl groups include, but are not limited to, phenyl,naphthyl, azulenyl, and anthracenyl.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁-C₆ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁-C₆alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃-C₆carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O, N or S, and inpreferred five membered monocyclic heterocyclyls, the heteroatom(s) areselected from one or two heteroatoms selected from O, N, or S. Examplesof heterocyclyl rings include, but are not limited to, azepinyl,acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇carbocyclyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, and 3-10 memberedheterocyclyl, as defined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, and3-10 membered heterocyclyl, as defined herein. A non-limiting exampleincludes carboxyl (i.e., —C(═O)OH).

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇carbocyclyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, and 3-10 memberedheterocyclyl, as defined herein.

A “sulfino” group refers to a “—S(═O)OH” group.

A “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as definedherein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(b) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as definedherein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl, 5-10membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as definedherein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl, 5-10 memberedheteroaryl, and 3-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

An “aminoalkyl” group refers to an amino group connected via an alkylenegroup.

An “alkoxyalkyl” group refers to an alkoxy group connected via analkylene group, such as a “C₂-C₈ alkoxyalkyl” and the like.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substituents independently selected fromC₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇carbocyclyl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 3-10membered heterocyclyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 3-10 memberedheterocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), aryl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heteroaryl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheteroaryl(C₁-C₆)alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo, —CN,hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether), aryloxy,sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃), halo(C₁-C₆)alkoxy(e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino, amino(C₁-C₆)alkyl,nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl,cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl,—SO₃H, sulfino, —OSO₂C₁₋₄alkyl, and oxo (═O). Wherever a group isdescribed as “optionally substituted” that group can be substituted withthe above substituents.

The term “hydroxy” as used herein refers to a —OH group.

The term “cyano” group as used herein refers to a “—CN” group.

The term “azido” as used herein refers to a —N₃ group.

As used herein, a “nucleotide” includes a nitrogen containingheterocyclic base, a sugar, and one or more phosphate groups. They aremonomeric units of a nucleic acid sequence. In RNA, the sugar is aribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl groupthat is present in ribose. The nitrogen containing heterocyclic base canbe purine or pyrimidine base. Purine bases include adenine (A) andguanine (G), and modified derivatives or analogs thereof. Pyrimidinebases include cytosine (C), thymine (T), and uracil (U), and modifiedderivatives or analogs thereof. The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine.

As used herein, a “nucleoside” is structurally similar to a nucleotide,but is missing the phosphate moieties. An example of a nucleosideanalogue would be one in which the label is linked to the base and thereis no phosphate group attached to the sugar molecule. The term“nucleoside” is used herein in its ordinary sense as understood by thoseskilled in the art. Examples include, but are not limited to, aribonucleoside comprising a ribose moiety and a deoxyribonucleosidecomprising a deoxyribose moiety. A modified pentose moiety is a pentosemoiety in which an oxygen atom has been replaced with a carbon and/or acarbon has been replaced with a sulfur or an oxygen atom. A “nucleoside”is a monomer that can have a substituted base and/or sugar moiety.Additionally, a nucleoside can be incorporated into larger DNA and/orRNA polymers and oligomers.

The term “purine base” is used herein in its ordinary sense asunderstood by those skilled in the art, and includes its tautomers.Similarly, the term “pyrimidine base” is used herein in its ordinarysense as understood by those skilled in the art, and includes itstautomers. A non-limiting list of optionally substituted purine-basesincludes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine,7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acidand isoguanine. Examples of pyrimidine bases include, but are notlimited to, cytosine, thymine, uracil, 5,6-dihydrouracil and5-alkylcytosine (e.g., 5-methylcytosine).

As used herein, when an oligonucleotide or polynucleotide is describedas “comprising” a nucleoside or nucleotide described herein, it meansthat the nucleoside or nucleotide described herein forms a covalent bondwith the oligonucleotide or polynucleotide. Similarly, when a nucleosideor nucleotide is described as part of an oligonucleotide orpolynucleotide, such as “incorporated into” an oligonucleotide orpolynucleotide, it means that the nucleoside or nucleotide describedherein forms a covalent bond with the oligonucleotide or polynucleotide.In some such embodiments, the covalent bond is formed between a 3′hydroxy group of the oligonucleotide or polynucleotide with the 5′phosphate group of a nucleotide described herein as a phosphodiesterbond between the 3′ carbon atom of the oligonucleotide or polynucleotideand the 5′ carbon atom of the nucleotide.

As used herein, “derivative” or “analogue” means a synthetic nucleotideor nucleoside derivative having modified base moieties and/or modifiedsugar moieties. Such derivatives and analogs are discussed in, e.g.,Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al.,Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprisemodified phosphodiester linkages, including phosphorothioate,phosphorodithioate, alkyl-phosphonate, phosphoranilidate andphosphoramidate linkages. “Derivative”, “analog” and “modified” as usedherein, may be used interchangeably, and are encompassed by the terms“nucleotide” and “nucleoside” defined herein.

As used herein, the term “phosphate” is used in its ordinary sense asunderstood by those skilled in the art, and includes its protonatedforms (for example,

As used herein, the terms “monophosphate,” “diphosphate,” and“triphosphate” are used in their ordinary sense as understood by thoseskilled in the art, and include protonated forms.

The terms “protecting group” and “protecting groups” as used hereinrefer to any atom or group of atoms that is added to a molecule in orderto prevent existing groups in the molecule from undergoing unwantedchemical reactions. Sometimes, “protecting group” and “blocking group”can be used interchangeably.

As used herein, the prefixes “photo” or “photo-” mean relating to lightor electromagnetic radiation. The term can encompass all or part of theelectromagnetic spectrum including, but not limited to, one or more ofthe ranges commonly known as the radio, microwave, infrared, visible,ultraviolet, X-ray or gamma ray parts of the spectrum. The part of thespectrum can be one that is blocked by a metal region of a surface suchas those metals set forth herein. Alternatively, or additionally, thepart of the spectrum can be one that passes through an interstitialregion of a surface such as a region made of glass, plastic, silica, orother material set forth herein. In particular embodiments, radiationcan be used that is capable of passing through a metal. Alternatively,or additionally, radiation can be used that is masked by glass, plastic,silica, or other material set forth herein.

As used herein, the term “phasing” refers to a phenomenon in SB S thatis caused by incomplete removal of the 3′ terminators and fluorophores,and failure to complete the incorporation of a portion of DNA strandswithin clusters by polymerases at a given sequencing cycle. Pre-phasingis caused by the incorporation of nucleotides without effective 3′terminators, wherein the incorporation event goes 1 cycle ahead due to atermination failure. Phasing and pre-phasing cause the measured signalintensities for a specific cycle to consist of the signal from thecurrent cycle as well as noise from the preceding and following cycles.As the number of cycles increases, the fraction of sequences per clusteraffected by phasing and pre-phasing increases, hampering theidentification of the correct base. Pre-phasing can be caused by thepresence of a trace amount of unprotected or unblocked 3′-OH nucleotidesduring sequencing by synthesis (SBS). The unprotected 3′-OH nucleotidescould be generated during the manufacturing processes or possibly duringthe storage and reagent handling processes. Accordingly, the discoveryof nucleotide analogues which decrease the incidence of pre-phasing issurprising and provides a great advantage in SBS applications overexisting nucleotide analogues. For example, the nucleotide analoguesprovided can result in faster SBS cycle time, lower phasing andpre-phasing values, and longer sequencing read lengths.

3′-Hydroxy Acetal Blocking Groups

Some embodiments of the present disclosure relate to a nucleotide ornucleoside molecule comprising a ribose or deoxyribose having aremovable 3′-OH protecting or blocking group forming a structure

covalently attached to the 3′-carbon atom, wherein:

each R^(1a) and R^(1b) is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, cyano, halogen, optionally substitutedphenyl, or optionally substituted aralkyl;

each R^(2a) and R^(2b) is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl,cyano, or halogen; alternatively R^(1a) and R^(2a) together with theatoms to which they are attached form an optionally substituted five toeight membered heterocyclyl group;

R³ is H, optionally substituted C₂-C₆ alkenyl, optionally substitutedC₃-C₇ cycloalkenyl, optionally substituted C₂-C₆ alkynyl, or optionallysubstituted (C₁-C₆ alkylene)Si(R⁴)₃; and

each R⁴ is independently H, C₁-C₆ alkyl, or optionally substitutedC₆-C₁₀ aryl; provided that when each R^(1a), R^(1b), R^(2a) and R^(2b)is H, then R³ is not H.

Some further embodiments of the present disclosure relate to a compoundhaving the structure of Formula (I):

wherein R′ is H, monophosphate, di-phosphate, tri-phosphate,thiophosphate, a phosphate ester analog, —O— attached to a reactivephosphorous containing group, or —O— protected by a protecting group; R″is H or OH; B is a nucleobase; each of R^(1a), R^(1b), R^(2a), R^(2b)and R³ is defined above. In some further embodiment, B is

In some further embodiments, the nucleobase is covalently bounded to adetectable label (e.g., a fluorescent dye), optionally through a linker,for example, B is

In some such embodiments, R′ is triphosphate. In some such embodiment,R″ is H.

In some embodiments of the acetal blocking group described herein, atleast one of R^(1a) and R^(1b) is H. In some such embodiments, eachR^(1a) and R^(1b) is H. In some other embodiments, at least one ofR^(1a) and R^(1b) is C₁-C₆ alkyl, for example, methyl, ethyl, isopropylor t-butyl. In some embodiments, each of R^(2a) and R^(2b) isindependently H, halogen or C₁-C₆ alkyl. In some such embodiments, atleast one of R^(2a) and R^(2b) is H or C₁-C₆ alkyl. In some suchembodiment, each R^(2a) and R^(2b) is H. In some such embodiments, eachR^(2a) and R^(2b) is C₁-C₆ alkyl, for example methyl, ethyl, isopropylor t-butyl. In one embodiment, each R^(2a) and R^(2b) is methyl. In somesuch embodiments, each R^(2a) and R^(2b) is independently C₁-C₆ alkyl orhalogen. In some such embodiments, R^(2a) is H, and R^(2b) is halogen orC₁-C₆ alkyl.

In some embodiments of the acetal blocking group described herein, R³ isoptionally substituted C₂-C₆ alkenyl. In some such embodiments, R³ isC₂-C₆ alkenyl (for example, vinyl, propenyl) optionally substituted withone or more substituents independently selected from the groupconsisting of halogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, and combinationsthereof. In some further embodiments, R³ is

In some other embodiments, R³ is optionally substituted C₂-C₆ alkynyl.In some such embodiments, R³ is C₂-C₆ alkynyl (e.g., ethynyl, propynyl)optionally substituted with one or more substituents independentlyselected from the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl and combinations thereof. In one embodiment, R³ is optionallysubstituted ethynyl

In some other embodiments, R³ is optionally substituted (C₁-C₆alkylene)Si(R⁴)₃. In some such embodiments, at least one of R⁴ is C₁₋₄alkyl. In some further embodiments, each one of R⁴ is C₁-C₄ alkyl, forexample, methyl, ethyl, isopropyl or t-butyl. In one embodiment, R³ is—(CH₂)—SiMe₃. In some alternative embodiments, R³ is C₁-C₆ alkyl.

In some alternative embodiments, R^(1a) and R^(2a) together with theatoms to which they are attached form a five to seven memberedheterocyclyl. In some such embodiments, R^(1a) and R^(2a) together withthe atoms to which they are attached form a six membered heterocyclyl.In some such embodiments, the six membered heterocyclyl group has thestructure

In some further embodiments, at least one of each R^(1b), R^(2b) and R³is H. In some other embodiments, at least one of each R^(1b), R^(2b) andR³ is C₁-C₆ alkyl. In one embodiment, each R^(1b), R^(2b) and R³ is H.

In some further embodiments, the compound of Formula (I) is alsorepresented by Formula (Ia):

where each R^(2c) and R^(2d) is independently H, halogen (e.g., fluoro,chloro), C₁-C₆ alkyl (e.g., methyl, ethyl, or isopropyl), or C₁-C₆haloalkyl (e.g., —CHF₂, —CH₂F, or —CF₃). In some such embodiments, oneof R^(1a) and R^(1b) is H. In some such embodiments, each R^(1a) andR^(1b) is H. In some other embodiments, at least one of R^(1a) andR^(1b) is C₁-C₆ alkyl, for example, methyl, ethyl, isopropyl or t-butyl.In some embodiments, each of R^(2a) and R^(2b) is independently H,halogen or C₁-C₆ alkyl. In some such embodiment, each R^(2a) and R^(2b)is H. In some such embodiments, each of R^(2c) and R^(2d) isindependently H, halogen or C₁-C₆ alkyl. In some such embodiments, eachR^(2c) and R^(2d) is C₁-C₆ alkyl, for example methyl, ethyl, isopropylor t-butyl. In one embodiment, each R^(2c) and R^(2d) is methyl. In somesuch embodiments, each R^(2c) and R^(2d) is independently halogen. Insome such embodiments, R^(2c) is H, and R^(2d) is H, halogen (fluoro,chloro) or C₁-C₆ alkyl (e.g., methyl, ethyl, isopropyl or t-butyl). Infurther embodiments, each R^(1a) and R^(1b) is H; R^(2a) is H; R^(2b) isH, halogen or methyl; R^(2c) is H; and R^(2d) is H, halogen, methyl,ethyl, isopropyl or t-butyl.

Non-limiting embodiments of the blocking groups described hereinincluding those having the structure selected from the group consistingof:

covalently attached to the 3′-carbon of the ribose or deoxyribose.

3′-Hydroxy Thiocarbamate Blocking Groups

Some additional embodiments of the present disclosure relate to anucleoside or nucleotide comprising a ribose or deoxyribose having aremovable 3′-OH blocking group forming a structure

covalently attached to the 3′-carbon atom, wherein:

each of R⁵ and R⁶ is independently H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ haloalkyl, C₂-C₈ alkoxyalkyl, optionally substituted—(CH₂)_(m)-phenyl, optionally substituted —(CH₂)_(n)-(5 or 6 memberedheteroaryl), optionally substituted —(CH₂)_(k)—C₃-C₇ carbocyclyl, oroptionally substituted —(CH₂)_(p)-(3 to 7 membered heterocyclyl);

alternatively, R⁵ and R⁶ together with the atoms to which they areattached form an optionally substituted five to seven memberedheterocyclyl;

each of —(CH₂)_(m)—, —(CH₂)_(n)—, —(CH₂)_(k)—, and —(CH₂)_(p)— isoptionally substituted; and

each of m, n, k, and p is independently 0, 1, 2, 3, or 4.

Some additional embodiments relate to a compound of Formula (II):

wherein R′ is H, monophosphate, di-phosphate, tri-phosphate,thiophosphate, a phosphate ester analog, —O— attached to a reactivephosphorous containing group, or —O— protected by a protecting group; R″is H or OH; B is a nucleobase; each of R⁵ and R⁶ is defined above. Insome further embodiment, B is

In some further embodiments, the nucleobase is covalently bounded to adetectable label (e.g., a fluorescent dye), optionally through a linker,for example, B is

In some such embodiments, R′ is triphosphate. In some such embodiment,R″ is H.

In some embodiments of the thiocarbamate blocking group describedherein, at least one of R⁵ and R⁶ is H. In some such embodiments, eachR⁵ and R⁶ is H. In some such embodiments, R⁵ is H and R⁶ is C₁-C₆ alkyl,for example, methyl, ethyl, isopropyl or t-butyl. In some suchembodiments, R⁵ is H and R⁶ is C₂-C₆ alkenyl (for example, vinyl orallyl) or C₂-C₆ alkynyl (for example, ethynyl or propynyl). In some suchembodiments, R⁵ is H and R² is optionally substituted —(CH₂)_(m)-phenyl,optionally substituted —(CH₂)_(n)-(5 or 6 membered heteroaryl),optionally substituted —(CH₂)_(k)—C₃-C₇ carbocyclyl, or optionallysubstituted —(CH₂)_(p)-(3 to 7 membered heterocyclyl). In some furtherembodiment, the C₃-C₇ carbocyclyl group may be a C₃-C₇ cycloalkyl orC₃-C₇ cycloalkenyl. The 3 to 7 membered heterocyclyl group may comprisezero or one double bond in the ring structure. In further embodiments,R⁵ is H and R⁶ is optionally substituted —(CH₂)_(m)-phenyl, optionallysubstituted —(CH₂)_(n)-6 membered heteroaryl, optionally substituted—(CH₂)_(k)—C₅ or C₆ carbocyclyl, or optionally substituted —(CH₂)_(p)-(5or 6 membered heterocyclyl). In some embodiments, m, n, k, or p is 0. Inother embodiments, m, n, k or p is 1 or 2. In some other embodiments, atleast one of R⁵ and R⁶ is C₁-C₆ alkyl, for example, methyl, ethyl,isopropyl or t-butyl. In some further embodiments, both R⁵ and R⁶ is areC₁-C₆ alkyl. In one embodiment, both R⁵ and R⁶ is are methyl.

In some alternative embodiments, R⁵ and R⁶ together with the atoms towhich they are attached form an optionally substituted five to sevenmembered heterocyclyl. In some such embodiments, R⁵ and R⁶ together withthe atoms to which they are attached form an optionally substitutedpiperidinyl.

Non-limiting embodiments of the 3′-O-thiocarbamate blocking groupsdescribed herein including those having the structure selected from thegroup consisting of:

covalently attached to the 3′-carbon of the ribose or deoxyribose.

Additional embodiments of the present disclosure relate to anoligonucleotide or a polynucleotide comprising a nucleoside ornucleotide described herein.

In any of the embodiments of the blocking groups described herein, whena group is described as “optionally substituted” it may be eitherunsubstituted or substituted.

In any embodiments of the nucleotides or nucleosides with the 3′ hydroxyblocking group described herein, the nucleoside or nucleotide may becovalently attached to a detectable label (for example, a fluorophore),optionally via a linker. The linker may be cleavable or non-cleavable.In some such embodiments, the detectable label (e.g., fluorophore) iscovalently attached to the nucleobase of the nucleoside or nucleotidevia a cleavable linker. In some other embodiments, the detectable label(e.g., fluorophore) is covalently attached to the 3′oxygen of thenucleoside or nucleotide via a cleavable linker. In some furtherembodiments, such cleavable linker may comprise an azido moiety or adisulfide moiety, an acetal moiety, or a thiocarbamate moiety. In someembodiments, the 3′ hydroxy blocking group and the cleavable linker (andthe attached label) may be removed under the same or substantially samechemical reaction conditions, for example, the blocking group and thedetectable label may be removed in a single chemical reaction. In otherembodiments, the blocking group and the detectable labeled are removedin two separate steps.

In some embodiments, the nucleotides or nucleosides described hereincomprises 2′ deoxyribose. In some further aspects, the 2′ deoxyribosecontains one, two or three phosphate groups at the 5′ position of thesugar ring. In some further aspect, the nucleotides described herein arenucleotide triphosphate.

In some embodiments, the 3′ blocked nucleotides or nucleosides describedherein provide superior stability in solution during storage, or reagenthandling during sequencing applications, compared to the samenucleotides or nucleosides protected with a standard 3′-OH blockinggroup disclosed in the prior art, for example, the 3′-O-azidomethylprotecting group. For example, the acetal or thiocarbamate blockinggroups disclosed herein may confer at least 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, 1500%, 2000%, 2500%, or 3000% improved stability compare toan azidomethyl protected 3′-OH at the same condition for the same periodof time, thereby reducing the pre-phasing values and resulting in longersequencing read lengths. In some embodiments, the stability is measuredat ambient temperature or a temperature below ambient temperature (suchas 4-10° C.). In other embodiments, the stability is measured at anelevated temperature, such as 40° C., 45° C., 50° C., 55° C., 60° C. or65° C. In some such embodiments, the stability is measured in solutionin a basic pH environment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0.In some such embodiments, the stability is measured with or without thepresence of an enzyme, such as a polymerase (e.g., a DNA polymerase), aterminal deoxynucleotidyl transferase, or a reverse transcriptase.

In some embodiments, the 3′ blocked nucleotides or nucleosides describedherein provide superior deblocking rate in solution during the chemicalcleavage step of the sequencing applications, compared to the samenucleotides or nucleosides protected with a standard 3′-OH blockinggroup disclosed in the prior art, for example, the 3′-O-azidomethylprotecting group. For example, the acetal or thiocarbamate blockinggroups disclosed herein may confer at least 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%,800%, 900%, 1000%, 1500%, or 2000% improved deblocking rate compare toan azidomethyl protected 3′-OH using the standard deblocking reagent(such as tris(hydroxypropyl)phosphine), thereby reducing the overalltime for a sequencing cycle. In some embodiments, the deblocking rate ismeasured at ambient temperature or a temperature below ambienttemperature (such as 4-10° C.). In other embodiments, the deblockingrate is measured at an elevated temperature, such as 40° C., 45° C., 50°C., 55° C., 60° C. or 65° C. In some such embodiments, the deblockingrate is measured in solution in a basic pH environment, e.g., at pH 9.0,9.2, 9.4, 9.6, 9.8. or 10.0. In some such embodiments, the molar ratioof the deblocking reagent to substrate (i.e., 3′ blocked nucleoside ornucleotide) is about 10:1, about 5:1, about 2:1 or about 1:1.

In some embodiments, a palladium deblocking reagent (e.g., Pd(0) is usedto remove the 3′ acetal blocking groups (e.g., AOM blocking group). Pdmay forms a chelation complex with the two oxygen atoms of the AOMgroup, as well as the double bond of the allyl group, allowing thedeblocking reagent in direct vicinity of the functionality to be removedand may result in accelerated deblocking rate.

Deprotection of the 3′-OH Blocking Groups

The 3′-acetal blocking groups described herein may be removed or cleavedunder various chemical conditions. For acetal blocking groups

that contain a vinyl or alkenyl moiety, non-limiting cleaving conditionincludes a Pd(II) complex, such as Pd(OAc)₂ or allylPd(II) chloridedimer, in the presence of a phosphine ligand, for exampletris(hydroxymethyl)phosphine (THMP), or tris(hydroxylpropyl)phosphine(THP or THPP). For those blocking groups containing an alkynyl group(e.g., an ethynyl), they may also be removed by a Pd(II) complex (e.g.,Pd(OAc)₂ or allyl Pd(II) chloride dimer) in the presence of a phosphineligand (e.g., THP or THMP).

Palladium Cleavage Reagents

In some embodiments, the acetal blocking group described herein may becleaved by a palladium catalyst. In some such embodiments, the Pdcatalyst is water soluble. In some such embodiments, is a Pd(0) complex(e.g., Tris(3,3′,3″-phosphinidynetris(benzenesulfonato)palladium(0)nonasodium salt nonahydrate). In some instances, the Pd(0) complex maybe generated in situ from reduction of a Pd(II) complex by reagents suchas alkenes, alcohols, amines, phosphines, or metal hydrides. Suitablepalladium sources include Na₂PdCl₄, Pd(CH₃CN)₂C₁₂, (PdCl(C₃H₅))₂,[Pd(C₃H₅)(THP)]C₁, [Pd(C₃H₅)(THP)₂]C₁, Pd(OAc)₂, Pd(Ph₃)₄, Pd(dba)₂,Pd(Acac)₂, PdCl₂(COD), and Pd(TFA)₂. In one such embodiment, the Pd(0)complex is generated in situ from Na₂PdCl₄. In another embodiment, thepalladium source is allyl palladium(II) chloride dimer [(PdCl(C₃H₅))₂].In some embodiments, the Pd(0) complex is generated in an aqueoussolution by mixing a Pd(II) complex with a phosphine. Suitablephosphines include water soluble phosphines, such astris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THMP),1,3,5-triaza-7-phosphaadamantane (PTA),bis(p-sulfonatophenyl)phenylphosphine dihydrate potassium salt,tris(carboxyethyl)phosphine (TCEP), andtriphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt.

In some embodiments, the Pd(0) is prepared by mixing a Pd(II) complex[(PdCl(C₃H₅))₂] with THP in situ. The molar ratio of the Pd(II) complexand the THP may be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or1:10. In some further embodiments, one or more reducing agents may beadded, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate).In some embodiments, the cleavage mixture may contain additional bufferreagents, such as a primary amine, a secondary amine, a tertiary amine,a carbonate salt, a phosphate salt, or a borate salt, or combinationsthereof. In some further embodiments, the buffer reagent comprisesethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine,sodium carbonate, sodium phosphate, sodium borate,2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine(TEMED), orN,N,N′,N′-tetraethylethylenediamine (TEEDA), or combinations thereof. Inone embodiment, the buffer reagent is DEEA. In another embodiment, thebuffer reagent contains one or more inorganic salts such as a carbonatesalt, a phosphate salt, or a borate salt, or combinations thereof. Inone embodiment, the inorganic salt is a sodium salt.

Alternatively, alkynyl moiety containing blocking groups may also becleaved in the presence of (NH₄)₂MoS₄. Other non-limiting cleavingcondition for alkynyl moiety includes a Cu(II) complex with THPTA ligand(tris(3-hydroxypropyltriazolylmethyl)amine), and ascorbate. Non-limitingcleaving conditions for blocking groups containing a six-memberedheterocycle (e.g., tetrahydropyran) include cyclodextrin or Ln(OTf)₃(lanthanide triflate). Non-limiting cleaving condition for blockinggroups containing an alkylsilane group (e.g., —CH₂SiMe₃) includes LiBF₄(lithium tetrafluoroborate). Other acetal blocking groups such as—O(CH₂)O—C₁-C₆ alkyl may be removed by LiBF₄ or Bi(OTf)₃ (bismuthtriflate). Non-limiting exemplary conditions for cleaving the describedvarious blocking groups are illustrated in Scheme 1 below.

The 3′-O-thiocarbamate blocking groups described herein may be removedor cleaved under various chemical conditions. Non-limiting exemplaryconditions for cleaving the thiocarbamate blocking groups describedherein include NaIO₄ and Oxone® (potassium peroxymonosulfate).

In addition, the azido group in —CH₂N₃ can be converted to an aminogroup by phosphine. Alternatively, the azido group in —CH₂N₃ may beconverted to an amino group by contacting such molecules with thethiols, in particular water-soluble thiols such as dithiothreitol (DTT).In one embodiment, the phosphine is THP.

Compatibility with Linearization

In order to maximize the throughput of nucleic acid sequencing reactionsit is advantageous to be able to sequence multiple template molecules inparallel. Parallel processing of multiple templates can be achieved withthe use of nucleic acid array technology. These arrays typically consistof a high-density matrix of polynucleotides immobilized onto a solidsupport material.

WO 98/44151 and WO 00/18957 both describe methods of nucleic acidamplification which allow amplification products to be immobilized on asolid support in order to form arrays comprised of clusters or“colonies” formed from a plurality of identical immobilizedpolynucleotide strands and a plurality of identical immobilizedcomplementary strands. Arrays of this type are referred to herein as“clustered arrays.” The nucleic acid molecules present in DNA colonieson the clustered arrays prepared according to these methods can providetemplates for sequencing reactions, for example as described in WO98/44152. The products of solid-phase amplification reactions such asthose described in WO 98/44151 and WO 00/18957 are so-called “bridged”structures formed by annealing of pairs of immobilized polynucleotidestrands and immobilized complementary strands, both strands beingattached to the solid support at the 5′ end. In order to provide moresuitable templates for nucleic acid sequencing, it is preferred toremove substantially all or at least a portion of one of the immobilizedstrands in the “bridged” structure in order to generate a template whichis at least partially single-stranded. The portion of the template whichis single-stranded will thus be available for hybridization to asequencing primer. The process of removing all or a portion of oneimmobilized strand in a “bridged” double-stranded nucleic acid structureis referred to as “linearization.” There are various ways forlinearization, including but not limited to enzymatic cleavage,photo-chemical cleavage, or chemical cleavage. Non-limiting examples oflinearization methods are disclosed in PCT Publication No. WO2007/010251, U.S. Patent Publication No. 2009/0088327, U.S. PatentPublication No. 2009/0118128, and U.S. Appl. 62/671,816, which areincorporated by reference in their entireties.

In some embodiments, the condition for deprotecting or removal of the3′-OH blocking groups is also compatible with the linearizationprocesses. In some further embodiments, the deprotection condition iscompatible with a chemical linearization process which comprises the useof a Pd complex and a phosphine, for example Pd(OAc)₂ and THP. In someembodiments, the Pd complex is a Pd(II) complex, which generates Pd(0)in situ in the presence of the phosphine.

Unless indicated otherwise, the reference to nucleotides is alsointended to be applicable to nucleosides.

Labeled Nucleotides

According to an aspect of the disclosure, the described 3′-OH blockednucleotide also comprises a detectable label and such nucleotide iscalled a labeled nucleotide. The label (e.g., a fluorescent dye) can beconjugated via an optional linker by a variety of means includinghydrophobic attraction, ionic attraction, and covalent attachment. Insome aspects, the dyes are conjugated to the substrate by covalentattachment. More particularly, the covalent attachment is by means of alinker group. In some instances, such labeled nucleotides are alsoreferred to as “modified nucleotides.”

Labeled nucleosides and nucleotides are useful for labelingpolynucleotides formed by enzymatic synthesis, such as, by way ofnon-limiting example, in PCR amplification, isothermal amplification,solid phase amplification, polynucleotide sequencing (e.g., solid phasesequencing), nick translation reactions and the like.

In some embodiments, the dye may be covalently attached tooligonucleotides or nucleotides via the nucleotide base. For example,the labeled nucleotide or oligonucleotide may have the label attached tothe C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base through a linker moiety.

Unless indicated otherwise, the reference to nucleotides is alsointended to be applicable to nucleosides. The present application willalso be further described with reference to DNA, although thedescription will also be applicable to RNA, PNA, and other nucleicacids, unless otherwise indicated.

Linkers

In some embodiments described herein, the purine or pyrimidine base ofthe nucleotide or nucleoside molecules described herein can be linked toa detectable label as described above. In some such embodiments, thelinkers used are cleavable. The use of a cleavable linker ensures thatthe label can, if required, be removed after detection, avoiding anyinterfering signal with any labeled nucleotide or nucleosideincorporated subsequently. In some embodiments, the cleavable linkercomprises an azido moiety, a —O—C₂-C₆ alkenyl moiety (e.g., —O-allyl), adisulfide moiety, an acetal moiety (same or similar to the 3′acetalblocking group described herein), or a thiocarbamate moiety (same orsimilar to the 3′acetal blocking group described herein).

In some other embodiments, the linkers used are non-cleavable. Since ineach instance where a labeled nucleotide of the invention isincorporated, no nucleotides need to be subsequently incorporated andthus the label need not be removed from the nucleotide.

Cleavable linkers are known in the art, and conventional chemistry canbe applied to attach a linker to a nucleotide base and a label. Thelinker can be cleaved by any suitable method, including exposure toacids, bases, nucleophiles, electrophiles, radicals, metals, reducing oroxidizing agents, light, temperature, enzymes etc. The linker asdiscussed herein may also be cleaved with the same catalyst used tocleave the 3′-O-blocking group bond. Suitable linkers can be adaptedfrom standard chemical protecting groups, as disclosed in Greene & Wuts,Protective Groups in Organic Synthesis, John Wiley & Sons. Furthersuitable cleavable linkers used in solid-phase synthesis are disclosedin Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

Where the detectable label is attached to the base, the linker can beattached at any position on the nucleotide base provided thatWatson-Crick base pairing can still be carried out. In the context ofpurine bases, it is preferred if the linker is attached via the7-position of the purine or the preferred deazapurine analogue, via an8-modified purine, via an N-6 modified adenosine or an N-2 modifiedguanine. For pyrimidines, attachment is preferably via the 5-position oncytosine, thymidine or uracil and the N-4 position on cytosine.

In some embodiments, the linker can comprise a spacer unit. The lengthof the linker is unimportant provided that the label is held asufficient distance from the nucleotide so as not to interfere with anyinteraction between the nucleotide and an enzyme, for example, apolymerase.

In some embodiments, the linker may consist of the similar functionalityas the 3′-OH protecting group. This will make the deprotection anddeprotecting process more efficient, as only a single treatment will berequired to remove both the label and the protecting group.

Use of the term “cleavable linker” is not meant to imply that the wholelinker is required to be removed. The cleavage site can be located at aposition on the linker that ensures that part of the linker remainsattached to the dye and/or substrate moiety after cleavage. Cleavablelinkers may be, by way of non-limiting example, electrophilicallycleavable linkers, nucleophilically cleavable linkers, photocleavablelinkers, cleavable under reductive conditions (for example disulfide orazide containing linkers), oxidative conditions, cleavable via use ofsafety-catch linkers and cleavable by elimination mechanisms. The use ofa cleavable linker to attach the dye compound to a substrate moietyensures that the label can, if required, be removed after detection,avoiding any interfering signal in downstream steps.

Useful linker groups may be found in PCT Publication No. WO2004/018493(herein incorporated by reference), examples of which include linkersthat may be cleaved using water-soluble phosphines or water-solubletransition metal catalysts formed from a transition metal and at leastpartially water-soluble ligands, for example, a Pd(II) complex and THP.In aqueous solution the latter form at least partially water-solubletransition metal complexes. Such cleavable linkers can be used toconnect bases of nucleotides to labels such as the dyes set forthherein.

Particular linkers include those disclosed in PCT Publication No.WO2004/018493 (herein incorporated by reference) such as those thatinclude moieties of the formulae:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C₁₋₁₀ substituted or unsubstituted alkyl group, Y is selectedfrom the group comprising O, S, NH and N(allyl), T is hydrogen or aC₁-C₁₀ substituted or unsubstituted alkyl group and * indicates wherethe moiety is connected to the remainder of the nucleotide ornucleoside). In some aspects, the linkers connect the bases ofnucleotides to labels such as, for example, the dye compounds describedherein.

Additional examples of linkers include those disclosed in U.S.Publication No. 2016/0040225 (herein incorporated by reference), such asthose include moieties of the formulae:

The linker moieties illustrated herein may comprise the whole or partiallinker structure between the nucleotides/nucleosides and the labels.

Additional examples of linkers (“L”) include moieties of the formula:

wherein B is a nucleobase; Z is —N₃ (azido), —O—C₁-C₆ alkyl, —O—C₂-C₆alkenyl, or —O—C₂-C₆ alkynyl; and Fl comprises a fluorescent label,which may contain additional linker structure. One of ordinary skill inthe art understands that label is covalently bounded to the linker byreacting a functional group of the label (e.g., carboxyl) with afunctional group of the linker (e.g., amino).

In particular embodiments, the length of the linker between afluorescent dye (fluorophore) and a guanine base can be altered, forexample, by introducing a polyethylene glycol spacer group, therebyincreasing the fluorescence intensity compared to the same fluorophoreattached to the guanine base through other linkages known in the art.Exemplary linkers and their properties are set forth in PCT PublicationNo. WO2007020457 (herein incorporated by reference). The design oflinkers, and especially their increased length, can allow improvementsin the brightness of fluorophores attached to the guanine bases ofguanosine nucleotides when incorporated into polynucleotides such asDNA. Thus, when the dye is for use in any method of analysis whichrequires detection of a fluorescent dye label attached to aguanine-containing nucleotide, it is advantageous if the linkercomprises a spacer group of formula —((CH₂)₂O)_(n)—, wherein n is aninteger between 2 and 50, as described in WO 2007/020457.

Nucleosides and nucleotides may be labeled at sites on the sugar ornucleobase. As known in the art, a “nucleotide” consists of anitrogenous base, a sugar, and one or more phosphate groups. In RNA, thesugar is ribose and in DNA is a deoxyribose, i.e., a sugar lacking ahydroxy group that is present in ribose. The nitrogenous base is aderivative of purine or pyrimidine. The purines are adenine (A) andguanine (G), and the pyrimidines are cytosine (C) and thymine (T) or inthe context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphateester of a nucleoside, with esterification occurring on the hydroxygroup attached to the C-3 or C-5 of the sugar. Nucleotides are usuallymono, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide but is missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogues areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analogue” means a compound or molecule whose core structure is the sameas, or closely resembles that of a parent compound but which has achemical or physical modification, such as, for example, a different oradditional side group, which allows the derivative nucleotide ornucleoside to be linked to another molecule. For example, the base maybe a deazapurine. In particular embodiments, the derivatives should becapable of undergoing Watson-Crick pairing. “Derivative” and “analogue”also include, for example, a synthetic nucleotide or nucleosidederivative having modified base moieties and/or modified sugar moieties.Such derivatives and analogues are discussed in, for example, Scheit,Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., ChemicalReviews 90:543-584, 1990. Nucleotide analogues can also comprisemodified phosphodiester linkages including phosphorothioate,phosphorodithioate, alkyl-phosphonate, phosphoranilidate,phosphoramidate linkages and the like.

A dye may be attached to any position on the nucleotide base, forexample, through a linker. In particular embodiments, Watson-Crick basepairing can still be carried out for the resulting analog. Particularnucleobase labeling sites include the C5 position of a pyrimidine baseor the C7 position of a 7-deaza purine base. As described above a linkergroup may be used to covalently attach a dye to the nucleoside ornucleotide.

In particular embodiments the labeled nucleoside or nucleotide may beenzymatically incorporable and enzymatically extendable. Accordingly, alinker moiety may be of sufficient length to connect the nucleotide tothe compound such that the compound does not significantly interferewith the overall binding and recognition of the nucleotide by a nucleicacid replication enzyme. Thus, the linker can also comprise a spacerunit. The spacer distances, for example, the nucleotide base from acleavage site or label.

Nucleosides or nucleotides labeled with the dyes described herein mayhave the formula:

where Dye is a dye compound; B is a nucleobase, such as, for exampleuracil, thymine, cytosine, adenine, guanine and the like; L is anoptional linker group which may or may not be present; R′ can be H,monophosphate, diphosphate, triphosphate, thiophosphate, a phosphateester analog, —O— attached to a reactive phosphorous containing group,or —O— protected by a blocking group; R′″ can be H, OH, aphosphoramidite, or a 3′-OH blocking group described herein, and R″ is Hor OH. Where R′″ is phosphoramidite, R′ is an acid-cleavable hydroxyprotecting group which allows subsequent monomer coupling underautomated synthesis conditions.

In a particular embodiment, the linker (between dye and nucleotide) andblocking group are both present and are separate moieties. In particularembodiments, the linker and blocking group are both cleavable undersubstantially similar conditions. Thus, deprotection and deblockingprocesses may be more efficient because only a single treatment will berequired to remove both the dye compound and the blocking group.However, in some embodiments a linker and blocking group need not becleavable under similar conditions, instead being individually cleavableunder distinct conditions.

The disclosure also encompasses polynucleotides incorporating dyecompounds. Such polynucleotides may be DNA or RNA comprised respectivelyof deoxyribonucleotides or ribonucleotides joined in phosphodiesterlinkage. Polynucleotides may comprise naturally occurring nucleotides,non-naturally occurring (or modified) nucleotides other than the labelednucleotides described herein or any combination thereof, in combinationwith at least one modified nucleotide (e.g., labeled with a dyecompound) as set forth herein. Polynucleotides according to thedisclosure may also include non-natural backbone linkages and/ornon-nucleotide chemical modifications. Chimeric structures comprised ofmixtures of ribonucleotides and deoxyribonucleotides comprising at leastone labeled nucleotide are also contemplated.

Non-limiting exemplary labeled nucleotides as described herein include:

wherein L represents a linker and R represents a sugar residue asdescribed above, or a sugar residue with the 5′ position substitutedwith one, two or three phosphates.

In some embodiments, non-limiting exemplary fluorescent dye conjugatesare shown below:

wherein PG stands for the 3′ hydroxy blocking groups described herein.In any embodiments of the labeled nucleotide described herein, thenucleotide is a nucleotide triphosphate.

Kits

The present disclosure also provides kits including one or more 3′blocked nucleosides and/or nucleotides described herein, for example,the 3′ blocked nucleotide of Formula (I), (Ia), or (II). Such kits willgenerally include at least one 3′ blocked nucleotide or nucleosidelabeled with a dye together with at least one further component. Thefurther component(s) may be one or more of the components identified ina method set forth herein or in the Examples section below. Somenon-limiting examples of components that can be combined into a kit ofthe present disclosure are set forth below.

In a particular embodiment, a kit can include at least one labeled 3′blocked nucleotide or nucleoside together with labeled or unlabelednucleotides or nucleosides. For example, nucleotides labeled with dyesmay be supplied in combination with unlabeled or native nucleotides,and/or with fluorescently labeled nucleotides or any combinationthereof. Combinations of nucleotides may be provided as separateindividual components (e.g., one nucleotide type per vessel or tube) oras nucleotide mixtures (e.g., two or more nucleotides mixed in the samevessel or tube).

Where kits comprise a plurality, particularly two, or three, or moreparticularly four, 3′ blocked nucleotides labeled with a dye compound,the different nucleotides may be labeled with different dye compounds,or one may be dark, with no dye compounds. Where the differentnucleotides are labeled with different dye compounds, it is a feature ofthe kits that the dye compounds are spectrally distinguishablefluorescent dyes. As used herein, the term “spectrally distinguishablefluorescent dyes” refers to fluorescent dyes that emit fluorescentenergy at wavelengths that can be distinguished by fluorescent detectionequipment (for example, a commercial capillary-based DNA sequencingplatform) when two or more such dyes are present in one sample. When twonucleotides labeled with fluorescent dye compounds are supplied in kitform, it is a feature of some embodiments that the spectrallydistinguishable fluorescent dyes can be excited at the same wavelength,such as, for example by the same laser. When four 3′ blocked nucleotides(A, C, T, and G) labeled with fluorescent dye compounds are supplied inkit form, it is a feature of some embodiments that two of the spectrallydistinguishable fluorescent dyes can both be excited at one wavelengthand the other two spectrally distinguishable dyes can both be excited atanother wavelength. Particular excitation wavelengths are 488 nm and 532nm.

In one embodiment, a kit includes a first 3′ blocked nucleotide labeledwith a first dye and a second nucleotide labeled with a second dyewherein the dyes have a difference in absorbance maximum of at least 10nm, particularly 20 nm to 50 nm. More particularly, the two dyecompounds have Stokes shifts of between 15-40 nm where “Stokes shift” isthe distance between the peak absorption and peak emission wavelengths.

In an alternative embodiment, the kits of the disclosure may contain 3′blocked nucleotides where the same base is labeled with two or moredifferent dyes. A first nucleotide (e.g., 3′ blocked T nucleotidetriphosphate or 3′ blocked G nucleotide triphosphate) may be labeledwith a first dye. A second nucleotide (e.g., 3′ blocked C nucleotidetriphosphate) may be labeled with a second spectrally distinct dye fromthe first dye, for example a “green” dye absorbing at less than 600 nm,and a “blue” dye absorbs at less than 500 nm, for example 400 nm to 500,in particular 450 nm to 460 nm). A third nucleotide (e.g., 3′ blocked Anucleotide triphosphate) may be labeled as a mixture of the first andthe second dyes, or a mixture of the first, the second and a third dyes,and the fourth nucleotide (e.g., 3′ blocked G nucleotide triphosphate or3′ blocked T nucleotide triphosphate) may be ‘dark’ and contain nolabel. In one example, the nucleotides 1-4 may be labeled ‘blue’,‘green’, ‘blue/green’, and dark. To simplify the instrumentationfurther, four nucleotides can be labeled with two dyes excited with asingle laser, and thus the labeling of nucleotides 1-4 may be ‘blue 1’,‘blue 2’, ‘blue 1/blue 2’, and dark.

In particular embodiments, the kits may contain four labeled 3′ blockednucleotides (e.g., A, C, T, G), where each type of nucleotide comprisesthe same 3′ blocking group and a fluorescent label, and wherein eachfluorescent label has a distinct fluorescence maximum and each of thefluorescent labels is distinguishable from the other three labels. Thekits may be such that two or more of the fluorescent labels have asimilar absorbance maximum but different Stokes shift. In some otherembodiments, one type of the nucleotide is unlabeled.

Although kits are exemplified herein in regard to configurations havingdifferent nucleotides that are labeled with different dye compounds, itwill be understood that kits can include 2, 3, 4 or more differentnucleotides that have the same dye compound. In some embodiments, thekit also includes an enzyme and a buffer appropriate for the action ofthe enzyme. In some such embodiments, the enzyme is a polymerase, aterminal deoxynucleotidyl transferase, or a reverse transcriptase. Inparticular embodiments, the enzyme is a DNA polymerase, such as DNApolymerase 812 (Pol 812) or DNA polymerase 1901 (Pol 1901). The aminoacid sequences of Pol 812 and Pol 1901 polymerases are described, forexample, in U.S. patent application Ser. No. 16/670,876, filed Oct. 31,2019, and Ser. No. 16/703,569, filed Dec. 4, 2019, both of which areincorporated by reference herein.

Other components to be included in such kits may include buffers and thelike. The nucleotides of the present disclosure, and other anynucleotide components including mixtures of different nucleotides, maybe provided in the kit in a concentrated form to be diluted prior touse. In such embodiments a suitable dilution buffer may also beincluded. Again, one or more of the components identified in a methodset forth herein can be included in a kit of the present disclosure.

Methods of Sequencing

Labeled nucleotides or nucleosides according to the present disclosuremay be used in any method of analysis such as method that includedetection of a fluorescent label attached to a nucleotide or nucleoside,whether on its own or incorporated into or associated with a largermolecular structure or conjugate. In this context the term “incorporatedinto a polynucleotide” can mean that the 5′ phosphate is joined inphosphodiester linkage to the 3′—OH group of a second (modified orunmodified) nucleotide, which may itself form part of a longerpolynucleotide chain. The 3′ end of a nucleotide set forth herein may ormay not be joined in phosphodiester linkage to the 5′ phosphate of afurther (modified or unmodified) nucleotide. Thus, in one non-limitingembodiment, the disclosure provides a method of detecting a nucleotideincorporated into a polynucleotide which comprises: (a) incorporating atleast one nucleotide of the disclosure into a polynucleotide and (b)detecting the nucleotide(s) incorporated into the polynucleotide bydetecting the fluorescent signal from the dye compound attached to saidnucleotide(s).

This method can include: a synthetic step (a) in which one or morenucleotides according to the disclosure are incorporated into apolynucleotide and a detection step (b) in which one or morenucleotide(s) incorporated into the polynucleotide are detected bydetecting or quantitatively measuring their fluorescence.

Some embodiments of the present application are directed to methods ofsequencing including: (a) incorporating at least one labeled nucleotideas described herein into a polynucleotide; and (b) detecting the labelednucleotide(s) incorporated into the polynucleotide by detecting thefluorescent signal from the new fluorescent dye attached to saidnucleotide(s).

Some embodiments of the present disclosure relate to a method fordetermining the sequence of a target single-stranded polynucleotide,comprising:

(a) incorporating a nucleotide comprising a 3′-OH blocking group and adetectable label as described herein into a copy polynucleotide strandcomplementary to at least a portion of the target polynucleotide strand;

(b) detecting the identity of the nucleotide incorporated into the copypolynucleotide strand; and

(c) chemically removing the label and the 3′-OH blocking group from thenucleotide incorporated into the copy polynucleotide strand.

In some embodiments, the sequencing method further comprises (d) washingthe chemically removed label and the 3′ blocking group away from thecopy polynucleotide strand. In some such embodiments, the 3′ blockinggroup and the detectable label are removed prior to introducing the nextcomplementary nucleotide. In some further embodiments, the 3′ blockinggroup and the detectable label are removed in a single step of chemicalreaction. In some embodiment, the washing step (d) also removeunincorporated nucleotides. In some further embodiments, a palladiumscavenger is also used in the washing step after chemical cleavage ofthe label and the 3′ blocking group.

In some embodiments, steps (a) to (d) is repeated until a sequence ofthe portion of the template polynucleotide strand is determined. In somesuch embodiments, steps (a) to (d) is repeated at least 50 times, atleast 75 times, at least 100 times, at least 150 times, at least 200times, at least 250 times, or at least 300 times.

In some embodiments, the label and the 3′ blocking group are removed intwo separate chemical reactions. In some such embodiments, removing thelabel from the nucleotide incorporated into the copy polynucleotidestrand comprises contacting the copy strand including the incorporatednucleotide with a first cleavage solution. In some such embodiment, thefirst cleavage solution contains a phosphine, such as atrialkylphosphine. None-limiting examples of trialkylphosphines includetris(hydroxypropyl)phosphine (THP), tris-(2-carboxyethyl)phosphine(TCEP), tris(hydroxymethyl)phosphine (THMP), ortris(hydroxyethyl)phosphine (THEP). In one embodiment, the firstcleavage solution contains THP. In some such embodiments, removing the3′ blocking group from the nucleotide incorporated into the copypolynucleotide strand comprises contacting the copy strand including theincorporated nucleotide with a second cleavage solution. In some suchembodiments, the second cleavage solution contains a palladium (Pd)catalyst. In some further embodiments, the Pd catalyst is a Pd(0)catalyst. In some such embodiments, the Pd(0) is prepared by mixing aPd(II) complex [(PdCl(C₃H₅))₂] with THP in situ. The molar ratio of thePd(II) complex and the THP may be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, or 1:10. In one embodiment the molar ration of Pd:THP is 1:5.In some further embodiments, one or more reducing agents may be added,such as ascorbic acid or a salt thereof (e.g., sodium ascorbate). Insome embodiments, the second cleavage solution may contain one or morebuffer reagents, such as a primary amine, a secondary amine, a tertiaryamine, a carbonate salt, a phosphate salt, or a borate salt, orcombinations thereof. In some further embodiments, the buffer reagentcomprises ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris),glycine, sodium carbonate, sodium phosphate, sodium borate,2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine(TEMED), orN,N,N′,N′-tetraethylethylenediamine (TEEDA), or combinations thereof. Inone embodiment, the buffer reagent is DEEA. In another embodiment, thebuffer reagent contains one or more inorganic salts such as a carbonatesalt, a phosphate salt, or a borate salt, or combinations thereof. Inone embodiment, the inorganic salt is a sodium salt. In some otherembodiments, the second cleavage solution contains NaIO₄ or Oxone®. Insome further embodiments, the 3′ blocked nucleotide contains a AOM groupand the second cleavage solution contains a palladium (Pd) catalyst andone or more buffer reagents described herein (e.g., a tertiary aminesuch as DEEA) and have pH of about 9.0 to about 10.0 (e.g., 9.6 or 9.8).

In some alternative embodiments, the label and the 3′-OH blocking groupare removed in a single chemical reaction. In some such embodiments, thelabel is attached to the nucleotide via a cleavage linker comprising thesame moiety as the 3′ blocking group, for example, both the linker andthe 3′ blocking group may comprise an acetal moiety

or a thiocarbamate moiety

as described herein. In some such embodiment, the single chemicalreaction is carried out in a cleavage solution containing a Pd catalystdescribed above.

In some further embodiments, the nucleotides used in the incorporationstep (a) are fully functionalized A, C, T and G nucleotide triphosphateeach contains a 3′blocking group described herein. In some suchembodiments, the nucleotides herein provide superior stability insolution during sequencing runs, compared to the same nucleotidesprotected with a standard 3′-O-azidomethyl blocking group. For example,the acetal or thiocarbamate blocking groups disclosed herein may conferat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or3000% improved stability compare to an azidomethyl protected 3′-OH atthe same condition for the same period of time, thereby reducing thepre-phasing values and resulting in longer sequencing read lengths. Insome embodiments, the stability is measured at ambient temperature or atemperature below ambient temperature (such as 4-10° C.). In otherembodiments, the stability is measured at an elevated temperature, suchas 40° C., 45° C., 50° C., 55° C., 60° C. or 65° C. In some suchembodiments, the stability is measured in solution in a basic pHenvironment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0. In somefurther embodiments, the pre-phasing value with the 3′ blockednucleotide described herein is less than about 0.25, 0.24, 0.23, 0.22,0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10,0.09, 0.08, 0.07, 0.06, or 0.05 after over 50, 100 or 150 cycles of SBS.In some further embodiments, the phasing value with the 3′ blockednucleotide is less than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19,0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07,0.06, or 0.05, after over 50, 100 or 150 cycles of SBS. In oneembodiment, each ffN contains the 3′-AOM group.

In some embodiments, the 3′ blocked nucleotides described herein providesuperior deblocking rate in solution during the chemical cleavage stepof the sequencing run, compared to the same nucleotides protected with astandard 3′-O-azidomethyl blocking group. For example, the acetal (e.g.,AOM) or thiocarbamate blocking groups disclosed herein may confer atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000%improved deblocking rate compare to an azidomethyl protected 3′-OH usingthe standard deblocking reagent (such as tris(hydroxypropyl)phosphine),thereby reducing the overall time for a sequencing cycle. In someembodiments, the deblocking time for each nucleotide is reduced by about5%, 10%, 20%, 30%, 40%, 50%, or 60%. For example, the deblocking timefor 3′-AOM and 3′-O-azidomethyl is about 4-5 seconds and about 9-10seconds respectively under certain chemical reaction condition. In someembodiments, the half life (t_(1/2)) of AOM blocking group is at least1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold faster than azidomethyl blockinggroup. In some such embodiment, t_(1/2) of AOM is about 1 minute whilet_(1/2) of azidomethyl is about 11 minutes. In some embodiments, thedeblocking rate is measured at ambient temperature or a temperaturebelow ambient temperature (such as 4-10° C.). In other embodiments, thedeblocking rate is measured at an elevated temperature, such as 40° C.,45° C., 50° C., 55° C., 60° C. or 65° C. In some such embodiments, thedeblocking rate is measured in solution in a basic pH environment, e.g.,at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0. In some such embodiments, themolar ratio of the deblocking reagent to substrate (i.e., 3′ blockednucleoside or nucleotide) is about 10:1, about 5:1, about 2:1, about1:1, about 1:2, about 1:5 or about 1:10. In one embodiment, each ffNcontains the 3′-AOM group.

In any embodiments of the methods described herein, the labelednucleotide is a nucleotide triphosphate. In any embodiments of themethod described herein, the target polynucleotide strand is attached toa solid support, such as a flow cell.

In one embodiment, at least one nucleotide is incorporated into apolynucleotide in the synthetic step by the action of a polymeraseenzyme. In some such embodiments, the polymerase may be DNA polymerasePol 812 or Pol 1901. However, other methods of joining nucleotides topolynucleotides, such as, for example, chemical oligonucleotidesynthesis or ligation of labeled oligonucleotides to unlabeledoligonucleotides, can be used. Therefore, the term “incorporating,” whenused in reference to a nucleotide and polynucleotide, can encompasspolynucleotide synthesis by chemical methods as well as enzymaticmethods.

In a specific embodiment, a synthetic step is carried out and mayoptionally comprise incubating a template polynucleotide strand with areaction mixture comprising labeled 3′ blocked nucleotides of thedisclosure. A polymerase can also be provided under conditions whichpermit formation of a phosphodiester linkage between a free 3′—OH groupon a polynucleotide strand annealed to the template polynucleotidestrand and a 5′ phosphate group on the nucleotide. Thus, a syntheticstep can include formation of a polynucleotide strand as directed bycomplementary base-pairing of nucleotides to a template strand.

In all embodiments of the methods, the detection step may be carried outwhile the polynucleotide strand into which the labeled nucleotides areincorporated is annealed to a template strand, or after a denaturationstep in which the two strands are separated. Further steps, for examplechemical or enzymatic reaction steps or purification steps, may beincluded between the synthetic step and the detection step. Inparticular, the target strand incorporating the labeled nucleotide(s)may be isolated or purified and then processed further or used in asubsequent analysis. By way of example, target polynucleotides labeledwith nucleotide(s) as described herein in a synthetic step may besubsequently used as labeled probes or primers. In other embodiments,the product of the synthetic step set forth herein may be subject tofurther reaction steps and, if desired, the product of these subsequentsteps purified or isolated.

Suitable conditions for the synthetic step will be well known to thosefamiliar with standard molecular biology techniques. In one embodiment,a synthetic step may be analogous to a standard primer extensionreaction using nucleotide precursors, including nucleotides as describedherein, to form an extended target strand complementary to the templatestrand in the presence of a suitable polymerase enzyme. In otherembodiments, the synthetic step may itself form part of an amplificationreaction producing a labeled double stranded amplification productcomprised of annealed complementary strands derived from copying of thetarget and template polynucleotide strands. Other exemplary syntheticsteps include nick translation, strand displacement polymerization,random primed DNA labeling, etc. A particularly useful polymerase enzymefor a synthetic step is one that is capable of catalyzing theincorporation of nucleotides as set forth herein. A variety of naturallyoccurring or modified polymerases can be used. By way of example, athermostable polymerase can be used for a synthetic reaction that iscarried out using thermocycling conditions, whereas a thermostablepolymerase may not be desired for isothermal primer extension reactions.Suitable thermostable polymerases which are capable of incorporating thenucleotides according to the disclosure include those described in WO2005/024010 or WO 06/120433, each of which is incorporated herein byreference. In synthetic reactions which are carried out at lowertemperatures such as 37° C., polymerase enzymes need not necessarily bethermostable polymerases, therefore the choice of polymerase will dependon a number of factors such as reaction temperature, pH,strand-displacing activity and the like.

In specific non-limiting embodiments, the disclosure encompasses methodsof nucleic acid sequencing, re-sequencing, whole genome sequencing,single nucleotide polymorphism scoring, any other application involvingthe detection of the labeled nucleotide or nucleoside set forth hereinwhen incorporated into a polynucleotide. Any of a variety of otherapplications benefitting the use of polynucleotides labeled with thenucleotides comprising fluorescent dyes can use labeled nucleotides ornucleosides with dyes set forth herein.

In a particular embodiment, the disclosure provides use of labelednucleotides according to the disclosure in a polynucleotidesequencing-by-synthesis (SBS) reaction. Sequencing-by-synthesisgenerally involves sequential addition of one or more nucleotides oroligonucleotides to a growing polynucleotide chain in the 5′ to 3′direction using a polymerase or ligase in order to form an extendedpolynucleotide chain complementary to the template nucleic acid to besequenced. The identity of the base present in one or more of the addednucleotide(s) can be determined in a detection or “imaging” step. Theidentity of the added base may be determined after each nucleotideincorporation step. The sequence of the template may then be inferredusing conventional Watson-Crick base-pairing rules. The use of thelabeled nucleotides set forth herein for determination of the identityof a single base may be useful, for example, in the scoring of singlenucleotide polymorphisms, and such single base extension reactions arewithin the scope of this disclosure.

In an embodiment of the present disclosure, the sequence of a templatepolynucleotide is determined by detecting the incorporation of one ormore 3′ blocked nucleotides described herein into a nascent strandcomplementary to the template polynucleotide to be sequenced through thedetection of fluorescent label(s) attached to the incorporatednucleotide(s). Sequencing of the template polynucleotide can be primedwith a suitable primer (or prepared as a hairpin construct which willcontain the primer as part of the hairpin), and the nascent chain isextended in a stepwise manner by addition of nucleotides to the 3′ endof the primer in a polymerase-catalyzed reaction.

In particular embodiments, each of the different nucleotidetriphosphates (A, T, G and C) may be labeled with a unique fluorophoreand also comprises a blocking group at the 3′ position to preventuncontrolled polymerization. Alternatively, one of the four nucleotidesmay be unlabeled (dark). The polymerase enzyme incorporates a nucleotideinto the nascent chain complementary to the template polynucleotide, andthe blocking group prevents further incorporation of nucleotides. Anyunincorporated nucleotides can be washed away and the fluorescent signalfrom each incorporated nucleotide can be “read” optically by suitablemeans, such as a charge-coupled device using laser excitation andsuitable emission filters. The 3′-blocking group and fluorescent dyecompounds can then be removed (deprotected) simultaneously orsequentially to expose the nascent chain for further nucleotideincorporation. Typically, the identity of the incorporated nucleotidewill be determined after each incorporation step, but this is notstrictly essential. Similarly, U.S. Pat. No. 5,302,509 (which isincorporated herein by reference) discloses a method to sequencepolynucleotides immobilized on a solid support.

The method, as exemplified above, utilizes the incorporation offluorescently labeled, 3′-blocked nucleotides A, G, C, and T into agrowing strand complementary to the immobilized polynucleotide, in thepresence of DNA polymerase. The polymerase incorporates a basecomplementary to the target polynucleotide but is prevented from furtheraddition by the 3′-blocking group. The label of the incorporatednucleotide can then be determined, and the blocking group removed bychemical cleavage to allow further polymerization to occur. The nucleicacid template to be sequenced in a sequencing-by-synthesis reaction maybe any polynucleotide that it is desired to sequence. The nucleic acidtemplate for a sequencing reaction will typically comprise a doublestranded region having a free 3′—OH group that serves as a primer orinitiation point for the addition of further nucleotides in thesequencing reaction. The region of the template to be sequenced willoverhang this free 3′—OH group on the complementary strand. Theoverhanging region of the template to be sequenced may be singlestranded but can be double-stranded, provided that a “nick is present”on the strand complementary to the template strand to be sequenced toprovide a free 3′—OH group for initiation of the sequencing reaction. Insuch embodiments, sequencing may proceed by strand displacement. Incertain embodiments, a primer bearing the free 3′—OH group may be addedas a separate component (e.g., a short oligonucleotide) that hybridizesto a single-stranded region of the template to be sequenced.Alternatively, the primer and the template strand to be sequenced mayeach form part of a partially self-complementary nucleic acid strandcapable of forming an intra-molecular duplex, such as for example ahairpin loop structure. Hairpin polynucleotides and methods by whichthey may be attached to solid supports are disclosed in PCT PublicationNos. WO 01/57248 and WO 2005/047301, each of which is incorporatedherein by reference. Nucleotides can be added successively to a growingprimer, resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the base which has been added may bedetermined, particularly but not necessarily after each nucleotideaddition, thus providing sequence information for the nucleic acidtemplate. Thus, a nucleotide is incorporated into a nucleic acid strand(or polynucleotide) by joining of the nucleotide to the free 3′—OH groupof the nucleic acid strand via formation of a phosphodiester linkagewith the 5′ phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid template may comprise naturally occurring and/ornon-naturally occurring nucleotides and natural or non-natural backbonelinkages, provided that these do not prevent copying of the template inthe sequencing reaction.

In certain embodiments, the nucleic acid template to be sequenced may beattached to a solid support via any suitable linkage method known in theart, for example via covalent attachment. In certain embodimentstemplate polynucleotides may be attached directly to a solid support(e.g., a silica-based support). However, in other embodiments of thedisclosure the surface of the solid support may be modified in some wayso as to allow either direct covalent attachment of templatepolynucleotides, or to immobilize the template polynucleotides through ahydrogel or polyelectrolyte multilayer, which may itself benon-covalently attached to the solid support.

Embodiments and Alternatives of Sequencing-by-Synthesis

Some embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891;6,258,568 and 6,274,320, the disclosures of which are incorporatedherein by reference in their entireties). In pyrosequencing, releasedPPi can be detected by being immediately converted to adenosinetriphosphate (ATP) by ATP sulfurase, and the level of ATP generated isdetected via luciferase-produced photons. The nucleic acids to besequenced can be attached to features in an array and the array can beimaged to capture the chemiluminescent signals that are produced due toincorporation of a nucleotides at the features of the array. An imagecan be obtained after the array is treated with a particular nucleotidetype (e.g. A, T, C or G). Images obtained after addition of eachnucleotide type will differ with regard to which features in the arrayare detected. These differences in the image reflect the differentsequence content of the features on the array. However, the relativelocations of each feature will remain unchanged in the images. Theimages can be stored, processed and analyzed using the methods set forthherein. For example, images obtained after treatment of the array witheach different nucleotide type can be handled in the same way asexemplified herein for images obtained from different detection channelsfor reversible terminator-based sequencing methods.

In another exemplary type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures ofwhich are incorporated herein by reference. This approach is beingcommercialized by Solexa (now Illumina, Inc.), and is also described inWO 91/06678 and WO 07/123,744, each of which is incorporated herein byreference. The availability of fluorescently-labeled terminators inwhich both the termination can be reversed, and the fluorescent labelcleaved facilitates efficient cyclic reversible termination (CRT)sequencing. Polymerases can also be co-engineered to efficientlyincorporate and extend from these modified nucleotides.

Preferably in reversible terminator-based sequencing embodiments, thelabels do not substantially inhibit extension under SBS reactionconditions. However, the detection labels can be removable, for example,by cleavage or degradation. Images can be captured followingincorporation of labels into arrayed nucleic acid features. Inparticular embodiments, each cycle involves simultaneous delivery offour different nucleotide types to the array and each nucleotide typehas a spectrally distinct label. Four images can then be obtained, eachusing a detection channel that is selective for one of the fourdifferent labels. Alternatively, different nucleotide types can be addedsequentially, and an image of the array can be obtained between eachaddition step. In such embodiments each image will show nucleic acidfeatures that have incorporated nucleotides of a particular type.Different features will be present or absent in the different images duethe different sequence content of each feature. However, the relativeposition of the features will remain unchanged in the images. Imagesobtained from such reversible terminator-SBS methods can be stored,processed and analyzed as set forth herein. Following the image capturestep, labels can be removed, and reversible terminator moieties can beremoved for subsequent cycles of nucleotide addition and detection.Removal of the labels after they have been detected in a particularcycle and prior to a subsequent cycle can provide the advantage ofreducing background signal and crosstalk between cycles. Examples ofuseful labels and removal methods are set forth below.

Some embodiments can utilize detection of four different nucleotidesusing fewer than four different labels. For example, SBS can beperformed utilizing methods and systems described in the incorporatedmaterials of U.S. Pub. No. 2013/0079232. As a first example, a pair ofnucleotide types can be detected at the same wavelength, butdistinguished based on a difference in intensity for one member of thepair compared to the other, or based on a change to one member of thepair (e.g. via chemical modification, photochemical modification orphysical modification) that causes apparent signal to appear ordisappear compared to the signal detected for the other member of thepair. As a second example, three of four different nucleotide types canbe detected under particular conditions while a fourth nucleotide typelacks a label that is detectable under those conditions, or is minimallydetected under those conditions (e.g., minimal detection due tobackground fluorescence, etc.). Incorporation of the first threenucleotide types into a nucleic acid can be determined based on presenceof their respective signals and incorporation of the fourth nucleotidetype into the nucleic acid can be determined based on absence or minimaldetection of any signal. As a third example, one nucleotide type caninclude label(s) that are detected in two different channels, whereasother nucleotide types are detected in no more than one of the channels.The aforementioned three exemplary configurations are not consideredmutually exclusive and can be used in various combinations. An exemplaryembodiment that combines all three examples, is a fluorescent-based SBSmethod that uses a first nucleotide type that is detected in a firstchannel (e.g. dATP having a label that is detected in the first channelwhen excited by a first excitation wavelength), a second nucleotide typethat is detected in a second channel (e.g. dCTP having a label that isdetected in the second channel when excited by a second excitationwavelength), a third nucleotide type that is detected in both the firstand the second channel (e.g. dTTP having at least one label that isdetected in both channels when excited by the first and/or secondexcitation wavelength) and a fourth nucleotide type that lacks a labelthat is not, or minimally, detected in either channel (e.g. dGTP havingno label).

Further, as described in the incorporated materials of U.S. Pub. No.2013/0079232, sequencing data can be obtained using a single channel. Insuch so-called one-dye sequencing approaches, the first nucleotide typeis labeled but the label is removed after the first image is generated,and the second nucleotide type is labeled only after a first image isgenerated. The third nucleotide type retains its label in both the firstand second images, and the fourth nucleotide type remains unlabeled inboth images.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate oligonucleotides andidentify the incorporation of such oligonucleotides. Theoligonucleotides typically have different labels that are correlatedwith the identity of a particular nucleotide in a sequence to which theoligonucleotides hybridize. As with other SBS methods, images can beobtained following treatment of an array of nucleic acid features withthe labeled sequencing reagents. Each image will show nucleic acidfeatures that have incorporated labels of a particular type. Differentfeatures will be present or absent in the different images due thedifferent sequence content of each feature, but the relative position ofthe features will remain unchanged in the images. Images obtained fromligation-based sequencing methods can be stored, processed and analyzedas set forth herein. Exemplary SBS systems and methods which can beutilized with the methods and systems described herein are described inU.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize nanopore sequencing (Deamer, D. W. &Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapidsequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D.Branton, “Characterization of nucleic acids by nanopore analysis”, Acc.Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin,and J. A. Golovchenko, “DNA molecules and configurations in asolid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), thedisclosures of which are incorporated herein by reference in theirentireties). In such embodiments, the target nucleic acid passes througha nanopore. The nanopore can be a synthetic pore or biological membraneprotein, such as α-hemolysin. As the target nucleic acid passes throughthe nanopore, each base-pair can be identified by measuring fluctuationsin the electrical conductance of the pore. (U.S. Pat. No. 7,001,792;Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing usingsolid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K.“Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “Asingle-molecule nanopore device detects DNA polymerase activity withsingle-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008),the disclosures of which are incorporated herein by reference in theirentireties). Data obtained from nanopore sequencing can be stored,processed and analyzed as set forth herein. In particular, the data canbe treated as an image in accordance with the exemplary treatment ofoptical images and other images that is set forth herein.

Some other embodiments of sequencing method involves the use the 3′blocked nucleotide described herein in nanoball sequencing technique,such as those described in U.S. Pat. No. 9,222,132, the disclosure ofwhich is incorporated by reference. Through the process of rollingcircle amplification (RCA), a large number of discrete DNA nanoballs maybe generated. The nanoball mixture is then distributed onto a patternedslide surface containing features that allow a single nanoball toassociate with each location. In DNA nanoball generation, DNA isfragmented and ligated to the first of four adapter sequences. Thetemplate is amplified, circularized and cleaved with a type IIendonuclease. A second set of adapters is added, followed byamplification, circularization and cleavage. This process is repeatedfor the remaining two adapters. The final product is a circular templatewith four adapters, each separated by a template sequence. Librarymolecules undergo a rolling circle amplification step, generating alarge mass of concatemers called DNA nanoballs, which are then depositedon a flow cell. Goodwin et al., “Coming of age: ten years ofnext-generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. Nucleotide incorporations can be detectedthrough fluorescence resonance energy transfer (FRET) interactionsbetween a fluorophore-bearing polymerase and γ-phosphate-labelednucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and7,211,414, both of which are incorporated herein by reference, ornucleotide incorporations can be detected with zero-mode waveguides asdescribed, for example, in U.S. Pat. No. 7,315,019, which isincorporated herein by reference, and using fluorescent nucleotideanalogs and engineered polymerases as described, for example, in U.S.Pat. No. 7,405,281 and U.S. Pub. No. 2008/0108082, both of which areincorporated herein by reference. The illumination can be restricted toa zeptoliter-scale volume around a surface-tethered polymerase such thatincorporation of fluorescently labeled nucleotides can be observed withlow background (Levene, M. J. et al. “Zero-mode waveguides forsingle-molecule analysis at high concentrations.” Science 299, 682-686(2003); Lundquist, P. M. et al. “Parallel confocal detection of singlemolecules in real time.” Opt. Lett. 33, 1026-1028 (2008); Korlach, J. etal. “Selective aluminum passivation for targeted immobilization ofsingle DNA polymerase molecules in zero-mode waveguide nano structures.”Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures ofwhich are incorporated herein by reference in their entireties). Imagesobtained from such methods can be stored, processed and analyzed as setforth herein.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in U.S. Pub. Nos. 2009/0026082;2009/0127589; 2010/0137143; and 2010/0282617, all of which areincorporated herein by reference. Methods set forth herein foramplifying target nucleic acids using kinetic exclusion can be readilyapplied to substrates used for detecting protons. More specifically,methods set forth herein can be used to produce clonal populations ofamplicons that are used to detect protons.

The above SBS methods can be advantageously carried out in multiplexformats such that multiple different target nucleic acids aremanipulated simultaneously. In particular embodiments, different targetnucleic acids can be treated in a common reaction vessel or on a surfaceof a particular substrate. This allows convenient delivery of sequencingreagents, removal of unreacted reagents and detection of incorporationevents in a multiplex manner. In embodiments using surface-bound targetnucleic acids, the target nucleic acids can be in an array format. In anarray format, the target nucleic acids can be typically bound to asurface in a spatially distinguishable manner. The target nucleic acidscan be bound by direct covalent attachment, attachment to a bead orother particle or binding to a polymerase or other molecule that isattached to the surface. The array can include a single copy of a targetnucleic acid at each site (also referred to as a feature) or multiplecopies having the same sequence can be present at each site or feature.Multiple copies can be produced by amplification methods such as, bridgeamplification or emulsion PCR as described in further detail below.

The methods set forth herein can use arrays having features at any of avariety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher.

An advantage of the methods set forth herein is that they provide forrapid and efficient detection of a plurality of target nucleic acid inparallel. Accordingly the present disclosure provides integrated systemscapable of preparing and detecting nucleic acids using techniques knownin the art such as those exemplified above. Thus, an integrated systemof the present disclosure can include fluidic components capable ofdelivering amplification reagents and/or sequencing reagents to one ormore immobilized DNA fragments, the system comprising components such aspumps, valves, reservoirs, fluidic lines and the like. A flow cell canbe configured and/or used in an integrated system for detection oftarget nucleic acids. Exemplary flow cells are described, for example,in U.S. Pub. No. 2010/0111768 and U.S. Ser. No. 13/273,666, each ofwhich is incorporated herein by reference. As exemplified for flowcells, one or more of the fluidic components of an integrated system canbe used for an amplification method and for a detection method. Taking anucleic acid sequencing embodiment as an example, one or more of thefluidic components of an integrated system can be used for anamplification method set forth herein and for the delivery of sequencingreagents in a sequencing method such as those exemplified above.Alternatively, an integrated system can include separate fluidic systemsto carry out amplification methods and to carry out detection methods.Examples of integrated sequencing systems that are capable of creatingamplified nucleic acids and also determining the sequence of the nucleicacids include, without limitation, the MiSeq™ platform (Illumina, Inc.,San Diego, Calif.) and devices described in U.S. Ser. No. 13/273,666,which is incorporated herein by reference.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in WO 00/06770(incorporated herein by reference), wherein polynucleotides areimmobilized on a glass support by reaction between a pendant epoxidegroup on the glass with an internal amino group on the polynucleotide.In addition, polynucleotides can be attached to a solid support byreaction of a sulfur-based nucleophile with the solid support, forexample, as described in WO 2005/047301 (incorporated herein byreference). A still further example of solid-supported templatepolynucleotides is where the template polynucleotides are attached tohydrogel supported upon silica-based or other solid supports, forexample, as described in WO 00/31148, WO 01/01143, WO 02/12566, WO03/014392, U.S. Pat. No. 6,465,178 and WO 00/53812, each of which isincorporated herein by reference.

A particular surface to which template polynucleotides may beimmobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels aredescribed in the references cited above and in WO 2005/065814, which isincorporated herein by reference. Specific hydrogels that may be usedinclude those described in WO 2005/065814 and U.S. Pub. No.2014/0079923. In one embodiment, the hydrogel is PAZAM(poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide)).

DNA template molecules can be attached to beads or microparticles, forexample, as described in U.S. Pat. No. 6,172,218 (which is incorporatedherein by reference). Attachment to beads or microparticles can beuseful for sequencing applications. Bead libraries can be prepared whereeach bead contains different DNA sequences. Exemplary libraries andmethods for their creation are described in Nature, 437, 376-380 (2005);Science, 309, 5741, 1728-1732 (2005), each of which is incorporatedherein by reference. Sequencing of arrays of such beads usingnucleotides set forth herein is within the scope of the disclosure.

Templates that are to be sequenced may form part of an “array” on asolid support, in which case the array may take any convenient form.Thus, the method of the disclosure is applicable to all types ofhigh-density arrays, including single-molecule arrays, clustered arrays,and bead arrays. Labeled nucleotides of the present disclosure may beused for sequencing templates on essentially any type of array,including but not limited to those formed by immobilization of nucleicacid molecules on a solid support.

However, labeled nucleotides of the disclosure are particularlyadvantageous in the context of sequencing of clustered arrays. Inclustered arrays, distinct regions on the array (often referred to assites, or features) comprise multiple polynucleotide template molecules.Generally, the multiple polynucleotide molecules are not individuallyresolvable by optical means and are instead detected as an ensemble.Depending on how the array is formed, each site on the array maycomprise multiple copies of one individual polynucleotide molecule(e.g., the site is homogenous for a particular single- ordouble-stranded nucleic acid species) or even multiple copies of a smallnumber of different polynucleotide molecules (e.g., multiple copies oftwo different nucleic acid species). Clustered arrays of nucleic acidmolecules may be produced using techniques generally known in the art.By way of example, WO 98/44151 and WO 00/18957, each of which isincorporated herein, describe methods of amplification of nucleic acidswherein both the template and amplification products remain immobilizedon a solid support in order to form arrays comprised of clusters or“colonies” of immobilized nucleic acid molecules. The nucleic acidmolecules present on the clustered arrays prepared according to thesemethods are suitable templates for sequencing using the nucleotideslabeled with dye compounds of the disclosure.

The labeled nucleotides of the present disclosure are also useful insequencing of templates on single molecule arrays. The term “singlemolecule array” or “SMA” as used herein refers to a population ofpolynucleotide molecules, distributed (or arrayed) over a solid support,wherein the spacing of any individual polynucleotide from all others ofthe population is such that it is possible to individually resolve theindividual polynucleotide molecules. The target nucleic acid moleculesimmobilized onto the surface of the solid support can thus be capable ofbeing resolved by optical means in some embodiments. This means that oneor more distinct signals, each representing one polynucleotide, willoccur within the resolvable area of the particular imaging device used.

Single molecule detection may be achieved wherein the spacing betweenadjacent polynucleotide molecules on an array is at least 100 nm, moreparticularly at least 250 nm, still more particularly at least 300 nm,even more particularly at least 350 nm. Thus, each molecule isindividually resolvable and detectable as a single molecule fluorescentpoint, and fluorescence from said single molecule fluorescent point alsoexhibits single step photobleaching.

The terms “individually resolved” and “individual resolution” are usedherein to specify that, when visualized, it is possible to distinguishone molecule on the array from its neighboring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules.The general features of single molecule arrays will be understood byreference to published applications WO 00/06770 and WO 01/57248, each ofwhich is incorporated herein by reference. Although one use of thenucleotides of the disclosure is in sequencing-by-synthesis reactions,the utility of the nucleotides is not limited to such methods. In fact,the nucleotides may be used advantageously in any sequencing methodologywhich requires detection of fluorescent labels attached to nucleotidesincorporated into a polynucleotide.

In particular, the labeled nucleotides of the disclosure may be used inautomated fluorescent sequencing protocols, particularly fluorescentdye-terminator cycle sequencing based on the chain terminationsequencing method of Sanger and co-workers. Such methods generally useenzymes and cycle sequencing to incorporate fluorescently labeleddideoxynucleotides in a primer extension sequencing reaction. So-calledSanger sequencing methods, and related protocols (Sanger-type), utilizerandomized chain termination with labeled dideoxynucleotides.

Thus, the present disclosure also encompasses labeled nucleotides whichare dideoxynucleotides lacking hydroxyl groups at both of the 3′ and 2′positions, such dideoxynucleotides being suitable for use in Sanger typesequencing methods and the like.

Labeled nucleotides of the present disclosure incorporating 3′ blockinggroups, it will be recognized, may also be of utility in Sanger methodsand related protocols since the same effect achieved by using dideoxynucleotides may be achieved by using nucleotides having 3′-OH blockinggroups: both prevent incorporation of subsequent nucleotides. Wherenucleotides according to the present disclosure, and having a 3′blocking group are to be used in Sanger-type sequencing methods it willbe appreciated that the dye compounds or detectable labels attached tothe nucleotides need not be connected via cleavable linkers, since ineach instance where a labeled nucleotide of the disclosure isincorporated; no nucleotides need to be subsequently incorporated andthus the label need not be removed from the nucleotide.

In any embodiments of the methods described herein, the nucleotide usedin the sequencing application is a 3′ blocked nucleotide describedherein, for example, the nucleotide of Formula (I), (Ia), or (II). Inany embodiments, the 3′ blocked nucleotide is a nucleotide triphosphate.

Examples

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1. Preparation of 3′-Acetal Blocked Nucleosides

In this example, various 3′-acetal protected T nucleoside were preparedaccording to Scheme 2.

Preparation of T1: Into an oven-dried nitrogen-purged 100 mL flask wasadded 5-iodo-2′-deoxyuridine (5.0 g, 14.12 mmol). This was co-evaporated3 times with 30 mL of pyridine then brought under Nitrogen. AnhydrousPyridine (25 mL) was added and the reaction stirred at room temperatureuntil a homogenous solution was obtained (˜15 minutes). The mixture wascooled to 0° C. in an ice-water bath and tert-butyldiphenylsilylchloride (4.04 mL, 15.5 mmol) was added slowly, dropwise with vigorousstirring (˜1 hour). The reaction was maintained at 0° C. for 8 hoursuntil all SM consumed by TLC. Saturated aqueous ammonium chloridesolution (˜15 mL) was added and the reaction allowed to warm to roomtemperature. The mixture was diluted with ethyl acetate (100 mL) andwashed with saturated aqueous ammonium chloride (200 mL). The organiclayer was separated, and the aqueous layer was extracted with ethylacetate (4×50 mL). The organic layers were combined, dried (MgSO₄) andconcentrated in vacuo to give ˜8 g clear yellow oil after removal ofresidual solvent under high vacuum. The crude product T1 was purified byflash-column chromatography on silica as a white crystalline solid.Yield is 6.94 g (83%). LC-MS (Electrospray negative) 591.08 [M−H]

Preparation of T2: Into an oven-dried, nitrogen-purged, brown, 500 mLthree-necked flask was added T1 (6.23 g, 10.5 mmol), copper (I) iodide(200 mg, 1.05 mmol) and bis(triphenylphosphine)palladium(II) dichloride(369 mg, 0.526 mmol) under nitrogen. The flask was protected from lightand anhydrous degassed DMF (200 mL) added. To this solution was added2,2,2-trifluoro-N-prop-2-ynyl-acetamide (4.74 g, 31.6 mmol), followed bydegassed triethylamine (2.92 mL, 21.0 mmol). The reaction was stirred atroom temperature under nitrogen for 6 hours when no further startingmaterial was observed by TLC analysis. Volatiles were removed in vacuo(˜15 mins) and DMF removed under high vacuum (˜1 hour) to brown residue.This was dissolved in ethyl acetate (200 mL) and extracted with 0.1MEDTA in water (2×200 mL). The aqueous layers were combined and furtherextracted with ethyl acetate (200 mL). The organic phases were combined,dried (MgSO₄) and volatiles removed in vacuo (˜30 min) and further driedunder high vacuum (˜1 hour) to give about 8 g of crude brown/yellow oil.The mixture was purified by flash-column chromatography on silica gel asan off-white solid. Yield: 6.0 g (85%). LC-MS (Electrospray negative)614.19 [M−H].

Preparation of T3: To an oven dried nitrogen purged 100 mL flaskcontaining starting nucleoside T2 (2.0 g, 3.25 mmol) under nitrogen wasadded anhydrous DMSO (6.9 ml, 97.5 mmol) in one portion at roomtemperature and stirred until a homogeneous solution was formed. Aceticacid (11.1 mL, 195 mmol) followed by acetic anhydride (15.1 mL, 162.09mmol) were both added dropwise (˜5 minutes each). The mixture was warmedto 50° C. and stirred until complete consumption of the startingnucleoside (˜5 hours) by TLC (EtOAc/Petroleum ether 3:2). The reactionwas then concentrated to half volume and cooled down with an ice bath toapproximately 0.5° C. Work up commenced by slow addition of cold (˜0.5°C.) NaHCO₃ (aq, sat.) (45 mL) and further stirring allowed until no morefizzing observed (˜15 min). The solution was allowed to warm to roomtemperature, then the aqueous extracted into EtOAc (3×100 mL). Combinedorganic layers were dried over MgSO₄, filtered and the volatilesevaporated under reduced pressure and further by high vacuum. Crudeproduct T3 was purified by flash chromatography on silica gel as anoff-white solid. Yield: 1.79 g (82%). LC-MS (Electrospray negative)674.20 [M−H]⁻.

Preparation of T4: To a solution of the starting nucleoside T3 (1.79 g,2.649 mmol) in anhydrous CH₂Cl₂ (50 mL) under N₂ was added cyclohexene(1.34 mL, 13.2 mmol). The mixture was cooled with an ice bath to 0° C.and distilled sulfuryl chloride (322 μL, 3.97 mmol) was slowly addeddropwise (˜20 min) under N₂, After stirring for 20 min at thattemperature TLC (EtOAc:petroleum ether=3:2 v/v) indicated the fullconsumption of the starting nucleoside. The chloride intermediate wasthen quenched by direct, dropwise addition of freshly distilled thecorresponding unsaturated alcohol (5 eq.) as shown in Scheme 3. Theresulting solution was stirred at room temperature for 2 hours followedby evaporation of the volatiles under reduced pressure. The oily residuewas partitioned between EtOAc:brine (3:2) (125 mL). The organic layerwas separated and the aqueous was further extracted into EtOAc (2×50mL). Combined organic extracts were dried over MgSO₄, filtered and thevolatiles evaporated under reduced pressure. The oily residue waspartitioned between EtOAc:brine (3:2) (125 mL). The organic layer wasseparated and the aqueous was further extracted into EtOAc (2×50 mL).Combined organic extracts were dried over MgSO₄, filtered and thevolatiles evaporated under reduced pressure. The crude products T4 waspurified by flash chromatography on silica gel to yield the finalproducts as a yellow oil. Yield: 1.20 g (69%) for AOM; 1.29 g (71%) forPrOM; 1.34 g (71%) for DPrOM.

3′-AOM: Yellow oil. LC-MS (Electrospray negative) [M−H] 684.24.

3′-PrOM: Yellow oil. LC-MS (Electrospray negative) [M−H] 682.22.

3′-DPrOM: Yellow oil. LC-MS (Electrospray negative) [M−H] 710.25.

Preparation of T5: The starting material T4 (1.04 g, 1.516 mmol) in a 50mL round bottom flask under nitrogen was added anhydrous THF (9 mL) atroom temperature. Then TBAF (1.0 M in THF, 1.7 mL, 1.70 mmol) was addeddropwise and the solution stirred until all SM consumed by TLC (˜2hours). The solution turned orange over the course of the reaction.Volatiles were removed in vacuo to give an orange residue which wasdissolved in EtOAc (100 mL) and separated with NaHCO₃ (sat. aq) (60 mL).The two layers were separated, and the aqueous layer was extracted withEtOAc (60 mL). The organic layers were combined, dried (MgSO₄),filtered, and evaporated to give the crude product as a yellow oil. Thecrude product was purified by flash chromatography on silica gel toyield a clear yellow oil. Yield: 637 mg (94%) for AOM; 526 mg (78%) forPrOM; 617 mg (86%) for DPrOM.

3′-AOM: clear yellow oil. LC-MS (Electrospray negative) [M−H] 446.12.

3′-PrOM: Clear yellow oil (526 mg 78%). LC-MS (Electrospray negative):[M−H] 444.10.

3′-DPrOM: Clear yellow oil (617 mg 86%). LC-MS (Electrospray negative):[M−H] 472.13.

In addition, two additional 3′ blocked T nucleosides (3′-eAOM T and3′-iAOM T) were prepared following the similar fashion as describedabove. 3′-iAOM T: LC-MS (ES): (negative ion) m/z 325.5 (M−H⁺), (positiveion) 327.3 (M+H⁺). 3′-eAOM T: LC-MS (ES): (positive ion) m/z 341.3(M+1H⁺).

Example 2. 3′-OH Blocking Group Stability Testing

In this example, the stability tests for 5′-mP 3′-AOM T nucleotide wasperformed side by side in an incorporation buffer solution with standard5′-mP 3′-O-azidomethyl T nucleotide.

Formulation of the Buffer Solution

1 mL of 0.1 mM of each 5′-monophosphate 3′-protected T nucleotide in asolution of 100 mM ethanolamine buffer (pH 9.8), 100 mM NaCl, and 2.5 mMEDTA, was incubated at 65° C. in a heating block for 2 weeks. At settime points, 40 μL aliquots were taken and analyzed by HPLC to determinethe percentage of blocked nucleotide remaining and the eventualformation of unblocked nucleotide.

The stability testing results relating to the 5′-monophosphate3′-protected nucleotides with AOM, PrOM, DPrOM acetal protecting groupsand the standard azidomethyl blocking group are illustrated in FIG. 1.It was observed that 3′ blocked nucleotide monophosphate with AOM, PrOMand DPrOM blocking groups offered over 30-50 fold improvement in thereduction of the deblocking rate in the solution. This experiment mimicshow the corresponding fully functionalized nucleotides (ffNs) wouldbehave when stored in an incorporation mix on the cartridge of asequencing device. The stability improvement offered by these acetalprotecting groups would also lead to a lower pre-phasing rate insequencing runs. Finally, it improves the shelf-life of theincorporation mix reagent.

Example 3. 3′-AOM Deblocking Testing

In this example, deblocking tests for 5′-mP 3′-AOM T and the standard5′-mP 3′-O-azidomethyl T nucleotide were performed individually in asolution unique to each blocking group. Conditions were formulated tomimic Illumina's standard deblock reagent as closely as possible, andfollow the same methodology. Concentrations of active deblock reagent,buffer, and nucleoside are kept the same across all tests, but theidentity of each component was unique. In this way, the observeddifference in rate between the individual deblocking chemistries cannotdue to the differences in concentration of formulation.

Standard Azidomethyl Deblocking Condition

Nucleotide: 5′-monophosphate 3′-O-azidomethyl T. Active Deblock reagent:tris(hydroxypropyl)phosphine (THP) (1M in 18 mΩ water). (Optional)Additive: Sodium ascorbate (0.1 mM in 18 mΩ water) final conc.=1 mM.Buffer: Ethanolamine pH 9.8 (2M in 18 mΩ water). Quenching reagent:H₂O₂.

AOM Deblocking Condition

Nucleotide: 5′-monophosphate 3′-O-azidomethyl T. A stock solution of3′-AOM T was diluted to 0.1 mM in a 100 mM ethanolamine buffer (pH 9.8),in a glass vial under nitrogen. A stock solution of sodium ascorbateadditive was added to a final concentration of 0.1 mM and the solutionstirred 5 minutes. To commence the assay, the deblock reagents(Pd/THP=1/5; sodium ascorbate; ethanolamine) were added, to a finalconcentration of 1 mM THP, to the stirring solution at room temperature.At specified time points, 40 μL aliquots were taken and quenched with 6μL of a 1:3 mixture of EDTA/H₂O₂ (0.025:0.075 M). HPLC analysis wasperformed by measuring the area of the starting nucleoside peak, the3′-OH peak, and any other nucleotide peaks that appear in the HPLCchromatogram. No other nucleotide-based side products were observed.

The comparative result is shown in FIG. 2A. It was observed that AOMoffered a 10-fold speed improvement in term of deblocking rate insolution compared to the standard azidomethyl blocking group. Thisexperiment serves the purpose of mimicking how the corresponding ffNswould behave in sequencing during the deblocking step. The substantialimprovement in the deblocking speed would allow for a flush-throughdeblocking step instead of the 10 to 20 second incubation time typicallyused in certain Illumina sequencing platforms. As a result, thedeblocking rate will have a significant impact on sequencing bysynthesis (SBS) cycle time.

Similar experimental conditions were used for the deblocking assay for3′-eAOM T and 3′-iAOM T. As a single alteration, Pd catalyst tosubstrate ratio was reduced to 5:1 in order to observe less distinctdifferences in the deblocking rate. 3′-AOM T was used as a reference andthe results are illustrated in FIG. 2B. These results showed that thedeblocking rate of eAOM and iAOM are 2 to 3 times slower than AOM atthis specific concentration of the Pd catalyst deblocking reagent. Itcan be expected that the difference in deblocking rates among thesubstituted version and unsubstituted version of the AOM blocking groupswould be smaller when Pd catalyst to substrate ratio is higher.

Example 4. Optimization of Palladium Cleavage Mix for Sequencing

The Pd/THP catalyst used in the deblocking reaction described in Example2 is very air sensitive. When exposed to air, it showed a substantialloss of activity. In this example, an oxidation stress assay wasdeveloped to assess air sensitivity of different formulations of thepalladium cleavage mix.

0.5 mL of Pd cleave mix were aliquoted in a 5 mL glass vial and leftopen to air for 3 hours at room temperature. The residual activity ofthe oxidized cleavage mix was assessed by measuring the cleavage of3′-AOM T as follows. A stock solution of 3′-AOM T was diluted to 0.1 mMin 100 mM cleavage mix buffer. A stock solution of sodium ascorbate wasadded to a final concentration of 1 mM, followed by the oxidizedcleavage mix to a final 1/20 dilution. After 1 hour, 40 μL of thesolution were immediately quenched with 10 μL of a 1:1 mixture ofEDTA/H₂O₂ (0.25:0.25 M) and analyzed by HPLC. In this experiment,various buffer reagents were screened, including: primary amines (suchas ethanolamine, Tris and glycine); tertiary amines (such as2-dimethyalaminomethanol ((DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine (TEMED) orN,N,N′,N′-tetraethylethylenediamine (TEEDA)); and various inorganicsalts (such as a borate salt, an carbonate salt, a phosphate salt). Itwas observed that inorganic buffer reagents (such as sodium borate,sodium carbonate, sodium phosphate) offered the best air stability andthe palladium complex retained high % activities. In addition, tertiaryamines also substantially improved the stability of the Pd cleavage mixas compared to primary amines.

Based on these findings, two palladium cleavage mix were prepared. In afirst example, a stock solution of 250 mM borate buffer aq. (pH 9.6, 20mL) was diluted with water (14 mL) before addition of a stock solutionof THP (1 M in 100 mM Tris, pH 9, 5 mL, 5.0 mmol) and of allylpalladium(II) chloride dimer (183 mg, 0.5 mmol). The mixture was vigorouslystirred for a few minutes at room temperature before addition of 1 Msodium ascorbate aq. (0.5 mL, 0.5 mmol), 5 M NaCl aq. (10 mL) and 10%v/v Tween20 (0.5 mL). In a second example, a stock solution of 2 M DEEAbuffer aq. (pH 9.6, 0.6 mL) was diluted with water (7.6 mL) beforeaddition of a stock solution of THP (1 M in 100 mM Tris, pH 9, 1.2 mL,1.2 mmol) and of solid allylpalladium (II) chloride dimer (43.9 mg, 0.12mmol). The mixture was vigorously stirred for a few minutes at roomtemperature before addition of 1 M sodium ascorbate aq. (0.12 mL, 0.12mmol), 5 M NaCl aq. (2.4 mL) and 10% v/v Tween20 (0.12 mL).

Example 5. Preparation of Fully Functionalized Nucleotides and Uses forSequencing Application

In this example, the preparation of various fully functionalizednucleotides (ffNs) with 3′-AOM blocking group are described in details.These ffNs were also used in the sequencing by synthesis application onIllumina MiniSeq® platform.

Synthesis of Intermediate AOM C2

Nucleoside C1 (0.5 g, 0.64 mmol) was dissolved in anhydrous DCM (12 mL)under N₂, and the mixture was cooled to 0° C. Cyclohexene (0.32 mL, 3.21mmol) was added, followed by dropwise SO₂Cl₂ (1.0 M in DCM, 1.27 mL,1.27 mmol). Additional cyclohexene (0.32 mL, 3.21 mmol) was added beforequickly transferring the reaction to a rotary evaporator to remove allthe volatiles under reduced pressure. The solid residue was additionallydried under high vacuum for 10 min before being dissolved in anhydrousDCM (5 mL) under N₂. The mixture was cooled to 0° C. and ice-cold allylalcohol (5 mL) was added dropwise. The reaction was stirred at 0° C. for2 h, before being quenched by addition of sat. NaHCO₃aq. (50 mL) and DCM(30 mL). The two phases were separated, and the aqueous layer wasextracted with EtOAc (2×50 mL). The organic layers were combined, driedover MgSO₄, filtered and the volatiles were evaporated under reducedpressure. The crude product was purified by flash chromatography onsilica gel using a EtOAc/petroleum ether to give AOM C2 as a white solid(264 mg, 52% yield). LC-MS (Electrospray negative): [M−H] 787, [M+Cl]823.

Synthesis of Intermediate AOM C3

AOM C2 (246 mg, 0.31 mmol) was dissolved in anhydrous THF (9.5 mL) underN₂ and the mixture was cooled to 0° C. Acetic acid (0.054 mL, 0.94 mmol)was added, followed by dropwise TBAF (1.0 M in THF, 5 wt. % water, 0.99mL, 0.94 mmol). The reaction was stirred at 0° C. for 5 h, before beingdiluted with EtOAc (20 mL) and then poured into 0.05 M HCl aq. (20 mL).The two layers were separated, and the aqueous layer was extracted withEtOAc (2×20 mL). The organic layers were combined, dried over MgSO₄,filtered and the volatiles were evaporated under reduced pressure. Thecrude product was purified by flash chromatography on silica gel using aDCM/EtOAc to give AOM C3 as a yellowish solid (114 mg, 66% yield). LC-MS(Electrospray negative): [M−H] 549, [M+H₂O—H]567, [M+Cl] 585,(Electrospray positive): [M+H] 551, [M+H₂O+H] 569.

Synthesis of Intermediate AOM C4

AOM C3 (0.114 g, 0.21 mmol), freshly activated 4 Å molecular sieves,proton sponge (0.066 g, 0.31 mmol) and a magnetic stirrer were placedunder N₂ and anhydrous trimethyl phosphate (1.0 mL) was added. Thereaction was cooled at −10° C. and freshly distilled POCl₃ (23 μL, 0.25mmol) was added dropwise. The reaction was stirred at −10° C. for 1hour. A solution of pyrophosphate as bis-tri-n-butylammonium salt (0.5 Min DMF, 1.7 mL, 0.85 mmol) and anhydrous tri-n-butyl amine (0.41 mL,1.74 mmol) were premixed and added to the ice-cold activated nucleosidesolution in one portion. The mixture was vigorously stirred for 5minutes at room temperature. The reaction mixture was poured into aseparate flask containing a vigorously stirred solution of 2 M TEAB aq.(˜10 mL). The reaction flask was rinsed with a small amount of H₂O andthe washings added into the 2 M TEAB solution. The combined mixture wasthen stirred at room temperature for 4 hours, after which the solventwas evaporated under reduced pressure. The residue was dissolved in NH₃aq. (35%, ˜10 mL) and stirred at room temperature overnight. Thereaction was concentrated under vacuum and purified by flashchromatography on DEAE-Sephadex. The product was further purified bypreparative HPLC to give pure AOM C4 (62 μmol, 30% yield, determined byUV-Vis spectrometry, λ_(max)=294 nm, ε=8600 M⁻¹ cm⁻¹). LC-MS(Electrospray negative): [M−H] 589.

Synthesis of 3′-AOM-ffC-LN3-SO7181

LN3-SO7181 (0.0205 mmol) was dissolved in anhydrous DMA (4 mL) under N₂.N,N-diisopropylethylamine (28.6 μL, 0.164 mmol) was added, followed byTSTU (0.1 M in DMA, 234 μL, 0.0234 mmol). The reaction was stirred underN₂ at room temperature for 1 hour. In the meantime, an aqueous solutionof AOM C4 (0.0101 mmol) was evaporated to dryness under reducedpressure, resuspended in 0.1 M TEAB aq. (400 μL) and added to theLN3-SO7181 solution. The reaction was stirred at RT for 17.5 hours andthen quenched with 0.1M TEAB aq. (4 mL). The crude product was purifiedby flash chromatography on DEAE-Sephadex. The product was furtherpurified by preparative HPLC to give pure 3′-AOM-ffC-LN3-SO7181 (6.81μmol, 67% yield, determined by UV-Vis spectrometry, λ_(max)=644 nm,E=200000 M⁻¹ cm⁻¹). LC-MS (Electrospray negative): [M−H] 1561, [M−2H]781, [M−3H] 520.

Synthesis of Intermediate AOM A2

Nucleoside A1 (716 mg, 0.95 mmol) was dissolved in 10 mL of anhydrousdichloromethane under N₂ atmosphere, cyclohexene (481 μL, 4.75 mmol) wasadded and the solution was cooled to approximately −15° C. Sulfurylchloride (distilled, 92 μL, 1.14 mmol) was added dropwise and thereaction was stirred for 20 minutes. After all the starting material hadbeen consumed, an extra portion of cyclohexene was added (481 μL, 4.75mmol) and the reaction was evaporated to dryness under reduced pressure.The residue was quickly purged with nitrogen, then allyl alcohol (5 mL,˜100 mmol) was added under stirring at 0° C. The reaction was stirred at0° C. for 1 hour, then quenched with 50 mL of saturated aq. NaHCO₃. Themixture was extracted with 2×100 mL of ethyl acetate. The pooled organicphases were washed with 100 mL of water and 100 mL of brine, then driedover MgSO₄, filtered and evaporated to dryness. The residue was purifiedby flash chromatography on silica gel using Petroleum ether/EtOAc. 60%yield (435 mg, 0.57 mmol). LC-MS (ES and CI): (positive ion) m/z 763(M+H⁺); (negative ion) m/z 761 (M−H⁺).

Synthesis of Intermediate AOM A3

Nucleoside AOM A2 (476 mg, 0.62 mmol) was dissolved in dry THF (5 mL)under N₂ atmosphere, then a solution of 1.0 M TBAF in THF (750 μL, 0.75mmol) was added. The solution was stirred at room temperature for 1.5hours. The solution was diluted with 50 mL of EtOAc, then washed with100 mL of NaH₂PO₄ sat. (pH=3), and with 100 mL of brine. The organicphase was dried over MgSO₄, filtered and evaporated to dryness. Theresidue was purified by flash chromatography on silica gel usingEtOAc/MeOH. 90% yield (292 mg, 0.55 mmol). LC-MS (ES and CI): (positiveion) m/z 525 (M+H⁺); (negative ion) m/z 523 (M−H⁺).

Synthesis of Intermediate AOM A4

Nucleoside AOM A3 (285 mg, 0.544 mmol,) was dried under reduced pressureover P₂O₅ for 18 hrs. Anhydrous triethyl phosphate (2 mL) and somefreshly activated 4 Å molecular sieves were added to it under nitrogen,then the reaction flask was cooled to 0° C. in an ice-bath. Freshlydistilled POCl₃ (61 μL, 0.65 mmol) was added drop-wise followed byProton Sponge® (175 mg, 0.816 mmol). After the addition, the reactionwas further stirred at 0° C. for 15 minutes. Then, a 0.5 M solution ofpyrophosphate as bis-tri-n-butylammonium salt (5.4 mL, 2.72 mmol) inanhydrous DMF was quickly added, followed immediately by tri-n-butylamine (540 μL, 2.3 mmol). The reaction was kept in the ice-water bathfor another 10 minutes, then quenched by pouring it into 1 M aqueoustriethylammonium bicarbonate (TEAB, 20 mL) and stirred at roomtemperature for 4 hours. All the solvents were evaporated under reducedpressure. A 35% aqueous solution of ammonia (20 mL) was added to theabove residue and the mixture was stirred at room temperature for atleast 5 hours. The solvents were then evaporated under reduced pressure.The crude product was purified firstly by ion-exchange chromatography onDEAE-Sephadex A25 (100 g). The column was eluted with a gradient ofaqueous triethylammonium bicarbonate. The fractions containing thetriphosphate were pooled and the solvent was evaporated to dryness underreduced pressure. The crude material was further purified by preparativescale HPLC using a YMC-Pack-Pro C18 column, eluting with 0.1 M TEAB andacetonitrile. Compound AOM A4 was obtained as triethylammonium salt. 56%yield (306 μmol). LC-MS (ES and CI): (negative ion) m/z 612 (M−H⁺);(positive ion) m/z 614 (M+H⁺), 715 (M+Et₃NH⁺).

General Procedure for ffA Synthesis

The dye-linker (0.020 mmol) was dissolved in 2 mL of anhydrousN,N′-dimethylacetamide (DMA). N,N-diisopropylethylamine (28.4 μL, 0.163mmol) was added, followed byN,N,N,N-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate as 0.1 Msolution in anhydrous DMA (TSTU, 232 μL, 0.023 mmol). The reaction wasstirred under nitrogen at room temperature for 1 hour. In the meantime,an aqueous solution of the triphosphate AOM A4 (0.01 mmol) wasevaporated to dryness under reduced pressure and resuspended in 200 μLof 0.1 M triethylammonium bicarbonate (TEAB) solution in water. Theactivated dye-linker solution was added to the triphosphate and thereaction was stirred at room temperature for 18 hours. The crude productwas purified firstly by ion-exchange chromatography on DEAE-Sephadex A25(25 g). The fractions containing the triphosphate were pooled and thesolvent was evaporated to dryness under reduced pressure. The crudematerial was further purified by preparative scale RP-HPLC using aYMC-Pack-Pro C18 column. 3′-AOM-ffA-LN3-NR7180A: 38% yield (3.8 μmol).LC-MS (ES): (negative ion) m/z 1459 (M−H⁺), 729 (M−2H⁺), 486 (M−3H⁺).3′-AOM-ffA-LN3-BL-NR550S0: 37% yield (3.7 μmol). LC-MS (ES): (negativeion) m/z 1771 (M−H⁺), 885 (M−2H⁺), 589 (M−3H⁺). 3′-AOMffA-LN3-BL-NR650C5: 51% yield (51 μmol). LC-MS (ES): (negative ion) m/z1917 (M−H⁺), 958 (M−2H⁺), 645 (M−3H⁺).

Synthesis of Intermediate AOM G4

Known nucleoside dG3 (100 mg, 0.143 mmol) was dissolved in 10 mL ofanhydrous dichloromethane under N₂ atmosphere, Cyclohexene (72 μL, 0.714mmol) was added and the solution cooled to −12° C. Sulfuryl chloride(distilled, (1M in DCM), 171 μL, 0.171 mmol) was added dropwise and thereaction was stirred for 10 min. An extra portion of cyclohexene (72 μL,0.714 mmol) was added and the reaction stirred for 30 min at −12° C. Thereaction was evaporated to dryness under reduced pressure, the residuewas purged with nitrogen, and ice cold, neat allyl alcohol (distilled,0.8 mL, 12 mmol) was added under stirring at −12° C. The reaction wasstirred at −12° C. for 60 mins, then quenched with 2 mL of saturated aq.NaHCO₃. The mixture was separated with ethyl acetate (2 mL), the aqueouslayer extracted with ethyl acetate. Combined organic phases were washedwith 4 mL of water and 4 mL of brine, dried over MgSO₄, filtered andevaporated to crude oil. The residue was purified by flashchromatography on silica gel to give AOM G4 as a clear oil. 36% yield(50.9 mg, 0.072 mmol). LC-MS (ES and CI): (positive ion) m/z 710 [M+H]⁺;(negative ion) m/z 708 [M−H]⁻.

Synthesis of Intermediate AOM G5

Nucleoside AOM-G4 (111 mg, 0.156 mmol) was dissolved in dry THF (5 mL)under N₂ atmosphere. Acetic acid (27 μL, 0.468 mmol) was added, followedby a solution of 1.0 M TBAF in THF (296 μL, 0.296 mmol). The solutionwas stirred at room temperature for 5 hours. The solution was dilutedwith 10 mL of EtOAc, washed with 10 mL of 0.05 M aq. HCl and organicseparated. The aqueous phase was extracted with ethyl acetate. Thecombined organic phases were dried over MgSO₄, filtered and evaporatedto dryness. The residue was purified by flash chromatography on silicagel to give AOM G5 as a white solid. 44% yield (32.4 mg, 0.068 mmol).LC-MS (ES and CI): (positive ion) m/z 472 [M+H]⁺; (negative ion) m/z 470[M−H]⁻.

Synthesis of 3′-AOM-pppG

Nucleoside AOM-G5 (79 mg, 0.168 mmol,) with freshly activated 4 Åmolecular sieves were dried under reduced pressure over P₂O₅ for 18 hrs.Proton Sponge® (175 mg, 0.816 mmol) and anhydrous triethyl phosphate(0.8 mL) was added under nitrogen and stirred at room temperature for 1hour. The reaction flask was cooled to 0° C. in an ice-bath, freshlydistilled POCl₃ (19 μL, 0.202 mmol) was added drop-wise and the reactionwas stirred at 0° C. for 15 minutes. Then, a 0.5 M solution ofpyrophosphate as bis-tri-n-butylammonium salt (1.68 mL, 0.84 mmol) inanhydrous DMF was quickly added, followed immediately by tri-n-butylamine (168 μL, 0.705 mmol). The reaction was removed from the ice/waterbath and stirred vigorously for 5 minutes, then quenched by pouring itinto 1 M aqueous triethylammonium bicarbonate (TEAB, 6 mL) and stirredat room temperature for 18 hours. All the solvents were evaporated underreduced pressure. The residue was dissolved in 35% aqueous ammoniasolution (10 mL) and was stirred at room temperature for at least 5hours. The solvents were then evaporated under reduced pressure andfurther co-evaporated with water. The crude product was purified firstlyby ion-exchange chromatography on DEAE-Sephadex A25 (50 g). The columnwas eluted with a linear gradient of aqueous triethylammonium. Thefractions containing the triphosphate were collected and the solvent wasevaporated to dryness under reduced pressure. The crude material wasfurther purified by preparative scale HPLC using a YMC-Pack-Pro C18column. 3′-AOM-pppG was obtained as triethylammonium salt. 24% yield(39.7 μmol). LC-MS (ES and CI): (negative ion) m/z 576 [M−H]⁻; (positiveion) m/z 578 [M+H]⁺.

3′-AOM-ffT-LN3-NR550S0 was synthesized in a similar fashion as describedin the preparation of the 3′-AOM ffA and ffC.

Synthesis of Intermediate T1

5-Iodo-2′-deoxyuridine (3 g, 8.4 mmol) and palladium (II) acetate (1.6g, 7.14 mmol) were dissolved in dry degassed DMF, thenN-allyltrifluoroacetamide (6.4 mL, 42 mmol) was added. The solution wasplaced under vacuum, then purged with nitrogen for 3 times, thendegassed triethylamine (2.3 mL, 16.8 mmol) was added. The solution washeated to 80° C. for 2 hours. The black mixture was cooled down to roomtemperature then diluted with 50 mL of methanol. Approximately 0.5 g ofactivated charcoal was added, and the solution was filtered on Celite,then evaporated under reduced pressure to afford a brown thick oil. Thiscrude was purified by chromatography on silica gel using a EtOAc/MeOH.Yield: (2.27 g, 5.99 mmol). LC-MS (ES and CI): (negative ion) m/z 378(M−H⁺).

Synthesis of Intermediate T2

5-[3-(2,2,2-trifluoroacetamido)-allyl]-2′-deoxyuridine (T1) (2.55 g,6.72 mmol) was dissolved in dry DMF. Imidazole (1.37 g, 20.1 mmol) wasadded followed by 4-(dimethylamino)pyridine (410 mg, 3.36 mmol). Thereaction was cooled to 0° C., then tert-butyl(chloro)diphenylsilane(1.92 mL, 7.39 mmol) was added slowly in 3 portions, 30 minutes apart.The reaction was stirred at 0° C. for 6 hours. The solvent was thenevaporated, and the residue resuspended in 200 mL of EtOAc and washedwith 2×200 mL aq. saturated NaHCO₃ and 200 mL of water, then 100 mL ofbrine. The organic phase was dried over MgSO₄, filtered and evaporatedto dryness. The crude was purified by flash chromatography on silicausing a DCM/EtOAc. 68% yield (2.806 g, 4.54 mmol). LC-MS (ES and CI):(positive ion) m/z 618 (M+H⁺); (negative ion) m/z 616 (M−H⁺).

Synthesis of Intermediate T3

5′-O-(tert-butyldiphenylsilyl)-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2′-deoxyuridine(T2) (2.8 g, 4.53 mmol) was dissolved in 10 mL of anhydrous DMSO (136mmol), then glacial acetic acid (16 mL, 272 mmol) and acetic anhydride(16 mL, 158 mmol) were added. The reaction was heated to 50° C. for 6hours then quenched with 200 mL of aq. saturated NaHCO₃. After thesolution stopped bubbling, it was extracted with 2×150 mL of EtOAc. Theorganic phases were pooled and washed with 2×200 mL of aq. saturatedNaHCO₃, 200 mL of water and 100 mL of brine. The organic phase was driedover MgSO₄, filtered and evaporated to dryness. The crude was purifiedby flash chromatography on silica using a DCM/EtOAc. 77% yield (2.375 g,3.51 mmol). LC-MS (ES and CI): (positive ion) m/z 678 (M+H⁺); (negativeion) m/z 676 (M−H⁺).

Synthesis of Intermediate T4

5′-O-(tert-butyldiphenylsilyl)-3′-O-methylthiomethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2′-deoxyuridine(T3) (310 mg, 0.45 mmol) was dissolved in 5 mL of anhydrousdichloromethane under N₂ atmosphere, cyclohexene (228 μL, 2.25 mmol) wasadded and the solution was cooled to approximately −15° C. Sulfurylchloride (distilled, 55 μL, 0.675 mmol) was added dropwise and thereaction was stirred for 20 minutes. After all the starting material hadbeen consumed, an extra portion of cyclohexene was added (228 μL, 2.25mmol) and the reaction was evaporated to dryness under reduced pressure.The residue was quickly purged with nitrogen, then ice-cold allylalcohol (2.5 mL) was added under stirring at 0° C. The reaction wasstirred at 0° C. for 35 minutes, then quenched with 25 mL of saturatedaq. NaHCO₃, then diluted further with 100 mL of saturated aq. NaHCO₃.The mixture was extracted with 2×50 mL of ethyl acetate. The pooledorganic phases were dried over MgSO₄, filtered and evaporated todryness. The residue was purified by flash chromatography on silica gelusing a DCM/EtOAc. 69% yield (214 mg, 0.311 mmol). LC-MS (ES and CI):(positive ion) m/z 688 (M+H⁺); (negative ion) m/z 686 (M−H⁺).

Synthesis of Intermediate T5

5′-O-(tert-butyldiphenylsilyl)-3′-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2′-deoxyuridine(T4) (210 mg, 0.305 mmol) was dissolved in dry THF (3 mL) under N₂atmosphere. A solution of 1.0 M TBAF in THF (367 μL, 0.367 mmol) wasadded. The solution was stirred at room temperature for 3 hours. Thesolution was diluted with 50 mL of EtOAc, then washed with 50 mL ofNaH₂PO₄ sat. (pH=3), and with 50 mL of water. The organic phase wasdried over MgSO₄, filtered and evaporated to dryness. The residue waspurified by flash chromatography on silica gel using DCM/EtOAc. 95%yield (130 mg, 0.289 mmol). LC-MS (ES and CI): (negative ion) m/z 448(M−H⁺), 484 (M+Cl⁻).

Synthesis of Intermediate T6

3′-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2′-deoxyuridine(T5) (120 mg, 0.267 mmol,) was dried under reduced pressure over P₂O₅for 18 hrs. Anhydrous triethyl phosphate (1 mL) and some freshlyactivated 4 Å molecular sieves were added to it under nitrogen, then thereaction flask was cooled to 0° C. Freshly distilled POCl₃ (30 μL, 0.32mmoles) was added drop-wise followed by Proton Sponge® (85 mg, 0.40mmol). After the addition, the reaction was further stirred at 0° C. for15 minutes. Then, a 0.5 M solution of pyrophosphate asbis-tri-n-butylammonium salt (2.7 mL, 1.33 mmol) in anhydrous DMF wasquickly added, followed immediately by tri-n-butyl amine (270 μL, 1.2mmol). The reaction was kept in the ice-water bath for another 10minutes, then quenched by pouring it into 1 M aqueous triethylammoniumbicarbonate (TEAB, 10 mL) and stirred at room temperature for 4 hours.All the solvents were evaporated under reduced pressure. A 35% aqueoussolution of ammonia (10 mL) was added to the above residue and themixture was stirred at room temperature for 18 hours. The solvents werethen evaporated under reduced pressure, the residue resuspended in 10 mLof 0.1 M TEAB and filtered. The filtrate was purified firstly byion-exchange chromatography on DEAE-Sephadex A25 (100 g). The column waseluted with aqueous triethylammonium bicarbonate (TEAB). The fractionscontaining the triphosphate were pooled and the solvent was evaporatedto dryness under reduced pressure. The crude material was furtherpurified by preparative scale HPLC using a YMC-Pack-Pro C18 column.Compound T6 was obtained as triethylammonium salt. 33% yield (89 μmol).LC-MS (ES and CI): (negative ion) m/z 592 (M−H⁺), 295 (M−2H⁺).

Synthesis of 3′-AOM-ffT-LN3′-NR550S0

The dried known compound LN3-NR550S0 (0.015 mmol) was dissolved inanhydrous DMA (2 mL) under N₂. N,N-diisopropylethylamine (17 μL, 0.1mmol) was added, followed by TSTU (0.1 M in DMA, 180 μL, 0.018 mmol).The reaction was stirred under N₂ at room temperature for 1 hour. In themeantime, an aqueous solution of T6 (0.01 mmol) was evaporated todryness under reduced pressure, resuspended in 0.1 M TEAB aq. (200 μL)and added to the LN3-NR550S0 solution. The reaction was stirred at RTfor 18 hours and then quenched with 0.1M TEAB aq. (4 mL). The crudeproduct was purified by flash chromatography on DEAE-Sephadex. Theproduct was further purified by preparative HPLC to give pure3′-AOM-ffT-LN3′-NR550SO. 67% yield (41 μmol, determined by UV-Visspectrometry, λ_(max)=550 nm, E=125000 M⁻¹cm⁻¹). LC-MS (ES): (negativeion) m/z 1521 (M−H⁺), 761 (M−2H⁺), 507 (M−3H⁺).

Sequencing-by-Synthesis Experiments

The ffNs were subsequently tested in sequencing using an IlluminaMiniSeq® instrument. With the exception of a new incorporation mixincluding these ffNs, all standard commercial reagents were used. Astandard 2×150 recipe was used. In addition to the standardsequencing-by-synthesis (SBS) protocols, a 5 seconds incubation in asolution of palladium cleavage mix (Pd:THP=1/5 in DEEA as described inExample 4) were added to unblock 3′-AOM.

In a first experiment, the following ffNs were used in the incorporationmix: 3′-AOM-ffT-LN3-NR550S0, 3′-AOM-ffA-LN3-BL-NR550S0,3′-AOM-ffA-LN3-BL-NR650C5, 3′-AOM-ffA-LN3-NR7180A, 3′-AOM-ffC-LN3-S7181,and 3′-AOM-pppG (dark G). and the sequencing result for Read 1 issummarized below.

Read % PF Phasing Pre-phasing ER 1 90.2% 0.798 0.159 3.12 % PF:Percentage of cluster passing filter after 26 cycles

In a second experiment, unlabeled 3′-AOM-pppT was synthesized similarlyto the preparation of 3′-AOM-pppG described above (LC-MS (ES): (negativeion) m/z 551 (M−H⁺)). It was used in presence of commercial greenffG-LN3-PEG12-ATTO532 (used on Illumina 4-channel systems) in sequencingand the same ffAs and ffC were used as those described in the firstexperiment above. The results are summarized below. There weresignificant improvements on phasing and pre-phasing values and no signaldecay was observed (FIG. 3A). In addition, error rates for both Read 1and Read 2 were also reduced.

Read Phasing Pre-phasing ER 1 0.173 0.046 1.21 2 0.175 0.057 1.99

In another experiment, an incorporation mix containing3′-AOM-ffT-LN3′-NR550S0, 3′-AOM-ffA-LN3-BL-NR550S0,3′-AOM-ffA-LN3-BL-NR650C5, 3′-AOM-ffA-LN3-NR7180A,3′-AOM-ffC-LN3-SO7181, and 3′-AOM-pppG (dark G) was used. Similarly toprevious runs, a 5 second incubation with a cleavage mixture containinga palladium catalyst (Pd/THP=1:10; 100 mM DEEA as described in Example4) was added to the standard SBS cycle. Standard MiniSeq® DNA polymerasewas used but at 2× incorporation time. No signal decay phenotype wasobserved (FIG. 3B). In addition, these sequencing results were comparedto commercial MiniSeq® runs (average of 3; N=3) using ffNs with thestandard azidomethyl blocking group. It was observed that the errorrates were nearly identical (FIG. 3C). The sequencing results aresummarized below.

Read Phasing Pre-phasing ER 1 0.121 0.063 0.40 2 0.129 0.062 0.57

In addition, the primary sequencing metrics for the ffNs with 3′-AOMblocking groups were compared to those produced by the standard MiniSeq®commercial kit including DNA polymerase Pol 812 and the comparativeresults are demonstrated in FIG. 4A. Very low pre-phasing was observeddue to the improved stability of the 3′-AOM-ffNs. However, phasing wasstill elevated even if 2× incorporation time was used.

In yet another experiment, a different DNA polymerase (Pol 1901) wasused instead of the DNA polymerase in the commercial MiniSeq® kits (Pol812). Pol 1901 allowed for standard 1× incorporation time in sequencinginstead of the 2× incorporation time described above. In addition,incubation in the Pd cleavage mixture was reduced by half compared tostandard run. This allowed for a 10% time saving on the complete SBSchemistry cycle. The sequencing metrics were significantly improved andexceeded the values obtained from the standard commercial kitscontaining 3′-O-azidomethyl blocking group (FIG. 4B).

3′ Blocking Group Stability Test in Sequencing

To demonstrate stability improvement of the ffNs with 3′-AOM, they werecompared side by side with standard MiniSeq® ffNs with 3′-O-azidomethylgroup. The two sets of ffNs were incubated at 45° C. for several days instandard incorporation mix formulations excluding only the DNApolymerase. For each time point, fresh polymerase was added to completethe incorporation mix directly prior loading on MiniSeq®. Sequencingconditions described previously were used. Pre-phasing % is a directindicator of the percentage of 3′OH-ffNs present in the mix thereforedirectly correlates to the stability of the 3′ block group. Pre-phasingvalues for both sets of ffNs were recorded and plotted (FIG. 5). At 45°C., it was observed that 3′-AOM containing ffNs appeared to be 6× morestable than standard ffNs with 3′-O-azidomethyl group. Sequencingmetrics also confirmed the trend observed during the stability assay insolution—3′-AOM block was significantly more stable than3′-O-azidomethyl group.

Example 6. Preparation of 3′-O-Thiocarbamate Blocked Nucleosides

In this example, various 3′-O-thiocarbamate protected T nucleoside wereprepared according to Scheme 8.

Preparation of T-7: Into an oven-dried nitrogen-purged 100 mL flask wasadded 5′-O-(4,4′-Dimethoxytrityl)thymidine (1.0 g, 1.836 mmol). This wasco-evaporated with anhydrous DMF (3×20 mL) and brought under nitrogen.Anhydrous DCM (9.2 mL) and 4-dimethylaminopyridine (224 mg, 0.184 mmol)were added and stirred at room temperature until a homogeneous solutionwas formed. Then 1,1′-thiocarbonyldiimidazole (360 mg, 2.02 mmol) wasadded quickly over a stream of nitrogen, the reaction resealed andstirred at room temperature for 2 hours until all starting material wasconsumed. The reaction mixture is filtered through a pad of silica geland the filter cake washed with EtOAc (10 mL). Volatiles were removed invacuo and the crude residue used without further purification.

Preparation of T-8: Compound T-7 from the previous step was usedimmediately after drying in vacuo. The residue was brought undernitrogen in a 25 mL round bottomed flash and dimethylamine (2 M in THF,7.3 mL, 14.6 mmol) was added and the reaction stirred for 2 hours untilall starting material was consumed according to TLC. All volatiles wereremoved in vacuo to a form a clear crude residue, which was purified byflash-column chromatography on silica gel to afford T-8 as a whitesolid. Yield: 1.15 g (99%). LC-MS (Electrospray negative) 630.23 [M−H].

Preparation of T-9: Starting nucleoside T-8 (320 mg, 0.504 mmol) wasdissolved in minimal acetonitrile in a 50 mL round bottomed flask inair. A solution of AcOH/H₂O 5:1 (12.5 mL:2.5 mL) was added in one go andthe reaction stirred at room temperature until all starting material wasconsumed (2-4 hours). Evaporation of all volatiles under vacuum andco-evaporating the residue in toluene (2×60 mL) provide crude product asan off white solid. The crude product was purified by flash columnchromatography to afford T-9 as a white solid. Yield: 123 mg (74%).LC-MS (Electrospray negative) [M−H] 328.10.

Following similar synthetic procedure using the corresponding MeNH₂ orNH₃, nucleosides with two other thiocarbamate protecting groups werealso prepared. The general reaction scheme is demonstrated below:

3′-O-Thiocarbamate Blocking Groups Stability Testing

The stability tests for 5′-mP 3′-DMTC T nucleotide was performed side byside in an incorporation buffer solution with standard 5′-mP3′-O-azidomethyl T nucleotide.

For both 5′-mP 3′-DMTC T and 5′-mP 3′-O-azidomethyl T, the finalsolution volume was 1 mL and the final concentrations of thecorresponding nucleotides were both 0.1 mM. Other components of theaqueous buffer solution include ethanolamine (EA), ethanolamine HCl,NaCl (100 mM), and EDTA (2.5 mM). The buffer solution has the followingconcentration: 0.5 M EA Buffer, 0.5 M NaCl, 0.01 M EDTA.

Stability Test Methodology

200 μL of 10× Buffer solution was added to a 1.7 mL polypropylene snaplock microtube and diluted with the correct volume of 18 mΩ water. Thecorresponding nucleoside was then added, the vial was sealed and mixedvia inversion, gentle stirring or pumping with a micropipette. A 40 μLaliquot was taken and analyzed by HPLC to act as a starting (or t=0)value. The vials are then placed in a pre-heated heating mantle set to65° C., covered with a thick layer of aluminum foil and left to heat forone month. 40 μL aliquots was taken periodically (week 1: once daily.Weeks 2-4: 1 every 2 days) and analyzed by HPLC to determine thepercentage starting material and the percentage deblocked (3′-OH)nucleotide in the samples. HPLC analysis was performed by measuring thearea of the stating nucleotide peak and the 3′OH peak. These values wereused to calculate a percentage of deblocked nucleotide which waspresented graphically and used to compare stability in an incorporationbuffer between samples. FIG. 6 illustrates the comparative results ofstability of three different thiocarbamate 3′ blocking nucleotides tothe nucleotide blocked with 3′-O-azidomethyl blocking group at 65° C. Itwas observed that while the nucleotide with 3′-O—C(═S)NH₂ or3′-O—C(═S)NHCH₃ was less stable than the nucleotide protected with thestandard 3′-O-azidomethyl group, the nucleotide with 3′-DMTC conferredimproved stability during the 9-day testing period. As such, DMTCdemonstrated superior stability over the standard azidomethyl blockinggroup.

Example 7. 3′-O-Thiocarbamate Blocking Group Deblocking Testing

In this example, deblocking or deblock tests for 5′-mP 3′-DMTC T and thestandard 5′-mP 3′-O-azidomethyl T nucleotide were performed individuallyin a solution unique to each blocking group. Conditions were formulatedto mimic Illumina's standard deblock reagent as closely as possible, andfollow the same methodology. Concentrations of active deblock reagent,buffer, and nucleoside are kept the same across all tests, but theidentity of each component was unique. In this way, the observeddifference in rate between the individual deblocking chemistries cannotdue to the differences in concentration of formulation.

Deblock Test General Methodology

Each reaction component was formulated individually as a concentratedstock in 18 mΩ water, stored appropriately, and aliquots combined in aspecific order given below. Reaction was commenced by addition of thepreformulated deblock reagent. Final Concentrations: nucleoside (0.1mM), active deblock reagent (1 mM), additive (specific to deblockreagent), buffer (100 mM). Final Volume: 2000 μL.

In a 3 mL glass vial was added the preformulated buffer solutionfollowed by preformulated additive solution. This was diluted with thecorrect volume of 18 mΩ water and stirred for 10 minutes. An aliquot ofnucleotide solution was then added and stirred for 5 minutes. A 40 μLaliquot was then taken, quenching reagent added and analyzed by HPLC asa reference (or t=0 min) peak. The deblock reagent was then added in onego to the stirring solution and timing was commenced. At specified timepoints, 40 μL aliquots were taken and quenched immediately with anappropriate quenching reagent, then analyzed by HPLC to determine theamount of deblocked nucleotide that has occurred at these specified timepoints. Results were plotted graphically and are used to compare deblockefficiency and efficacy.

DMTC Deblocking

Nucleotide: 5′-mP 3′-DMTC T. Active deblock reagent: NaIO₄ (0.1M in 18mΩ water) or Oxone® (0.1M in 18 mΩ water). Additive: none. Buffer forNaIO₄: pH 6.75 phosphate buffer (1M in 18 mΩ water). Buffer for Oxone®:pH 8.65 phosphate buffer (1M in 18 mΩ water). Quenching reagent: sodiumthiosulfate. The 3′-O-azidomethyl deblocking condition is the same asthose described in Example 3.

HPLC analysis was performed by measuring the area of the statingnucleoside peak, the 3′-OH peak, and any other nucleotide peaks thatappear in the HPLC chromatogram. These values were used to calculate apercentage of starting nucleotide and deblocked nucleotide which waspresented graphically and used to compare deblock rate, efficiency andefficacy between samples. The comparative result is shown in FIG. 7. Itwas observed that deblocking of DMTC with NaIO₄ was not efficient.However, the % of starting material remaining was significantly less forthe nucleotide with the DMTC blocking group when DMTC was cleaved withOxone®. In summary, DMTC has demonstrated a superior deblocking rate(with Oxone®) over the deblocking rate of the standard azidomethylblocking group.

1. A nucleoside or nucleotide comprising a ribose or deoxyribose havinga removable 3′-OH blocking group forming a structure

covalently attached to the 3′-carbon atom, wherein: each R^(1a) andR^(1b) is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy,C₁-C₆ haloalkoxy, cyano, halogen, optionally substituted phenyl, oroptionally substituted aralkyl; each R^(2a) and R^(2b) is independentlyH, C₁-C₆ alkyl, C₁-C₆ haloalkyl, cyano, or halogen; alternatively R^(1a)and R^(2a) together with the atoms to which they are attached form anoptionally substituted five to eight membered heterocyclyl group; R³ isH, optionally substituted C₂-C₆ alkenyl, optionally substituted C₃-C₇cycloalkenyl, optionally substituted C₂-C₆ alkynyl, or optionallysubstituted (C₁-C₆ alkylene)Si(R⁴)₃; and each R⁴ is independently H,C₁-C₆ alkyl, or optionally substituted C₆-C₁₀ aryl; provided that wheneach R^(1a), R^(1b), R^(2a) and R^(2b) is H, then R³ is not H.
 2. Thenucleoside or nucleotide of claim 1, wherein at least one of R^(1a) andR^(1b) is H.
 3. (canceled)
 4. The nucleoside or nucleotide of claim 1,wherein each of R^(2a) and R^(2b) is independently H, halogen or C₁-C₆alkyl.
 5. The nucleoside or nucleotide of claim 4, wherein each R^(2a)and R^(2b) is H.
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. Thenucleoside or nucleotide of claim 1, wherein R³ is C₂-C₆ alkynyloptionally substituted with one or more substituents independentlyselected from the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl and combinations thereof.
 10. The nucleoside or nucleotide ofclaim 9, wherein R³ is


11. The nucleoside or nucleotide of claim 1, wherein R³ is C₂-C₆ alkenyloptionally substituted with one or more substituents independentlyselected from the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆haloalkyl and combinations thereof.
 12. The nucleoside or nucleotide ofclaim 11, wherein R³ is


13. The nucleoside or nucleotide of claim 1, wherein R³ is optionallysubstituted (C₁-C₆ alkylene)Si(R⁴)₃ and wherein each R⁴ is C₁-C₆ alkyl.14. (canceled)
 15. The nucleoside or nucleotide of claim 1, whereinR^(1a) and R^(2a) together with the atoms to which they are attachedform a six membered heterocyclyl.
 16. (canceled)
 17. (canceled)
 18. Thenucleoside or nucleotide of claim 1, wherein the 3′-OH blocking groupcomprises the structure selected from the group consisting of:

covalently attached to the 3′-carbon of the ribose or deoxyribose.
 19. Anucleoside or nucleotide comprising a ribose or deoxyribose having aremovable 3′-OH blocking group forming a structure

covalently attached to the 3′-carbon atom, wherein: each of R⁵ and R⁶ isindependently H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆haloalkyl, C₂-C₈ alkoxyalkyl, optionally substituted —(CH₂)_(m)-phenyl,optionally substituted —(CH₂)_(n)-(5 or 6 membered heteroaryl),optionally substituted —(CH₂)_(k)—C₃-C₇ carbocyclyl, or optionallysubstituted —(CH₂)_(p)-(3 to 7 membered heterocyclyl); each of—(CH₂)_(m)—, —(CH₂)_(n)—, —(CH₂)_(k)—, and —(CH₂)_(p)— is optionallysubstituted; and each of m, n, k, and p is independently 0, 1, 2, 3, or4. 20.-25. (canceled)
 26. The nucleoside or nucleotide of claim 1,wherein the nucleoside or nucleotide is covalently attached to adetectable label, optionally via a cleavable linker.
 27. The nucleosideor nucleotide of claim 26, wherein the detectable label is covalentlyattached to a nucleobase of the nucleoside or nucleotide via a cleavablelinker.
 28. (canceled)
 29. The nucleoside or nucleotide of claim 27,wherein the cleavable linker comprises an azido moiety, a —O-allylmoiety, a disulfide moiety, an acetal moiety, or a thiocarbamate moiety.30. The nucleoside or nucleotide of claim 27, wherein the 3′-OH blockinggroup and the cleavable linker are removable under the same reactionconditions.
 31. The nucleoside or nucleotide of claim 1, comprising a 2′deoxyribonucleoside triphosphate.
 32. (canceled)
 33. An oligonucleotidecomprising the nucleotide of claim
 1. 34. A method of preparing agrowing polynucleotide complementary to a target single-strandedpolynucleotide in a sequencing reaction, comprising incorporating thenucleotide of claim 1 into a growing complementary polynucleotide,wherein the incorporation of the nucleotide prevents the introduction ofany subsequent nucleotide into the growing complementary polynucleotide.35. The method of claim 34, wherein the incorporation of the nucleotideis accomplished by a polymerase, a terminal deoxynucleotidyltransferase, or a reverse transcriptase.
 36. A method of determining thesequence of a target single-stranded polynucleotide, comprising: (a)incorporating the nucleotide of claim 26 into a copy polynucleotidestrand complementary to at least a portion of the target polynucleotidestrand; (b) identifying the nucleotide incorporated into the copypolynucleotide strand by detecting a signal from the detectable label;and (c) chemically removing the label and the 3′ blocking group from thenucleotide incorporated into the copy polynucleotide strand.
 37. Themethod of claim 36, further comprising (d) washing the chemicallyremoved label and the 3′ blocking group away from the copypolynucleotide strand.
 38. The method of claim 37, further comprisingrepeating steps (a) to (d) until a sequence of the portion of thetemplate polynucleotide strand is determined.
 39. The method of claim37, wherein the steps (a) to (d) is repeated at least 50 times.
 40. Themethod of claim 36, wherein the label and the 3′ blocking group from thenucleotide incorporated into the copy polynucleotide strand are removedin a single chemical reaction.
 41. The method of claim 40, wherein step(c) comprises contacting the incorporated nucleotide with a cleavagesolution comprising a palladium catalyst.
 42. The method of claim 36,wherein the label and the 3′ blocking group from the nucleotideincorporated into the copy polynucleotide strand are removed in twoseparate chemical reactions.
 43. The method of claim 41, wherein step(c) comprises contacting the incorporated nucleotide with a cleavagesolution comprising a phosphine, and a cleavage solution comprising apalladium catalyst.
 44. The method of claim 43, wherein the phosphine istris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine ortris(hydroxypropyl)phosphine.
 45. The method of claim 41, wherein thecleavage solution comprising the palladium catalyst further comprisesone or more buffer reagents selected from the group consisting of aprimary amine, a secondary amine, a tertiary amine, a carbonate salt, aphosphate salt, and a borate salt, and combinations thereof.
 46. Themethod of claim 45, wherein the buffer reagents are selected from thegroup consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane(Tris), glycine, a carbonate salt, a phosphate salt, a borate salt,2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine (TEMED), andN,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.47. A kit comprising one or more of the nucleosides or nucleotides ofclaim
 1. 48. (canceled)
 49. (canceled)
 50. (canceled)